Proteins, genes encoding them and method of using the same

ABSTRACT

The present invention provides, as a gene encoding an antigen recognized by a G-CSF inducing antibody, a gene encoding any one of the following proteins: (a) a protein having an amino acid sequence shown in SEQ ID NO; 2; (b) a protein having an amino acid sequence comprising a deletion, substitution, addition or insertion of one or several amino acids with respect to the amino acid sequence shown in SEQ ID NO: 2, and having a binding ability for an antibody which induces and secretes a granulocyte colony-stimulating factor or a fragment thereof: or (c) a protein having at least 50% or more homology with the amino acid sequence shown in SEQ ID NO: 2, and having a binding ability for an antibody capable of inducing and secreting a granulocyte colony-stimulating factor or a fragment thereof.

This application is the national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/JP01/08446 which has an Internationalfiling date of Sep. 27, 2001, which designated the United States ofAmerica.

TECHNICAL FIELD

The present invention relates to a protein having reactivity with anantibody capable of inducing and secreting a granulocytecolony-stimulating factor, a gene encoding the protein, and using methodthereof.

BACKGROUND ART

Granulocyte colony-stimulating factor (G-CSF) has a molecular weight ofapproximately 18,000 to 22,000. This factor is a glycoprotein, whichinduces the differentiation and proliferation of one type of a bloodcomponent leukocyte, neutrophil. This glycoprotein is comprised of 174amino acids (occasionally 178 amino acids) in the case of human, and itis composed of 178 amino acids in the case of mouse.

G-CSF affects to enhance the survival and function of matureneutrophils, the formation of erythroblasts by an erythropoietin, andthe formation of blast cell colonies by interleukin-3. Moreover. G-CSFpromotes (reinforces the function and increases the numbers of) bloodcells such as leukocytes, erythrocytes or thrombocytes. Examples of thecells generating G-CSF include macrophages, stromal cells, monocytes, Tlymphocytes, fibroblasts, vascular endothelial cells and others.

Administration of G-CSF as a therapuetic agent has an effect fortreatment of neutropenia as a side effect of an anticancer agent,neutropenia occurring after bone marrow transplantation, and aplasticanemia. However, when G-CSF is administered, it requires frequentadministration because of its low stability in the blood, and further,the administration route is limited to intravenous or subcutaneousadministration. Therefore, the use of G-CSF as a therapeutic agent ispainful for patients and imposing the burden to doctors. Moreover, ithas been reported that bone ache occurs as a side effect when G-CSF istherapeutically administered. Direct administration of macrophages orstroma cells, which produce G-CSF, has not been carried out, since thesecells happen to contain various types of proteins or substances and sounexpected side effects might occur.

As stated above, a method of differentiating and growing neutrophils bythe direct administration of G-CSF elicits bone ache as a side effectand requires frequent administration, thereby giving a certain pain andburden to both patients and doctors. Accordingly, the development ofanother treatment method is strongly required, but it has not beenestablished up till now.

Thus, the present inventors have intended that G-CSF is not directlyadministered but G-CSF is allowed to be produced and as a resultneutrophils are differentiated and grown, and they have previouslysucceeded in providing a G-CSF-inducing antibody (Japanese PatentApplication No. 9-266591 (Sep. 30, 1997) and Japanese Patent Laid-OpenNo. 11-106400 (Apr. 20, 1999). However, an antigen recognized by theG-CSF inducing antibody has not been clarified.

SUMMARY OF THE INVENTION

The present invention provides, as a gene encoding an antigen recognizedby an antibody capable of inducing and secreting G-CSF, a gene encodinga protein (a), (b) or (c) described as follows: (a) a protein having anamino acid sequence shown in SEQ. ID NO: 2; (b) a protein having anamino acid sequence comprising a deletion, substitution, addition orinsertion of one or several amino acids with respect to the amino acidsequence shown in SEQ ID NO: 2, and having a binding ability for anantibody capable of inducing and secreting a G-CSF or a fragmentthereof; or (c) a protein having at least 50% or more homology with theamino acid sequence shown in SEQ ID NO: 2, and having a binding abilityfor an antibody capable of inducing and secreting a G-CSF or a fragmentthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Western Blot Analysis of a 3-4H7 antibody and anti-MMRP19partial peptide antibodies (APAs).

FIG. 2: Hydropathy plot of an MMRP19 protein. The horizontal axis, thenumber of amino acid residues; the longitudinal axis, hydropathy index.The amino acid sequence from Nos. 99 to 128 of the center portion seemsto only represent the transmembrane (TM) domain of the MMRP19 protein.

FIG. 3: Reactivity to the RAW264.7 cells of antibodies, each of whichreacts against three types of partial peptide sequences of the MMRP19protein. This figure shows the results obtained by analyzing theorientation of the MMRP19 protein in the cell membrane of the RAW264.7cells by flow cytometry. The horizontal axis represents fluorescenceintensity and the vertical axis represents the cell number. Each ofAPA1, APA2 and APA3 represents an anti-peptide antibody.

FIG. 4: Induction activity of G-CSF gene by the 3-4H7 antibody or by theanti-MMRP19 partial peptide antibodies (APAs). The figure shows that theMMRP19 protein is associated to function the induction of expression ofa G-CSF gene. The horizontal axis represents the concentration ofanti-MMRP19 protein antibodies added, and the vertical axis representsluciferase activity which corresponds to the induction of the G-CSF geneexpression. The symbol ● represents the 3-4H7 antibody, the symbol ▪represents an anti-peptide antibody APA1, the symbol ♦ represents ananti-peptide antibody APA2, and the symbol ▴ represents an anti-peptideantibody APA3. Luciferase activity is shown as a relative value based ona respective control when a control value is defined as 1.

FIG. 5: Stimulating activity of G-CSF secretion by the 3-4H7 antibody(A) and LPS (B) in the RAW264.7 cells. The horizontal axis representsthe concentration of the antibody (μg/ml) or the concentration of theLPS (EU/ml), and the vertical axis represents promoting activity ofG-CSF secretion (%/maximum secretion).

FIG. 6: Gene induction and secretion promotion activity of G-CSF by the3-4H7 antibody (A) and LPS (B) in Pica-RAW264.7 cells. The horizontalaxes represent the concentration of the antibody (μg/ml) and theconcentration of the LPS (EU/ml), and the vertical axes represent theactivity of G-CSF gene induction or the promoting activity of G-CSFsecretion (%/maximum secretion). The symbol ● represents the activity ofG-CSF gene induction, and the symbol ◯ represents the promoting activityof G-CSF secretion.

FIG. 7: Secretion amount of G-CSF by the 3-4H7 antibody in the RAW264.7cells (n=6). The horizontal axis represents the concentrations of the3-4H7 antibodies (0, 1 and 10 μg/ml). The vertical axis represents thesecretion amount of G-CSF (pg/10⁴ cells).

FIG. 8: Secretion amount of G-CSF by the 3-4H7 antibody in Pica-RAW264.7cells (n=6). The horizontal axis represents the concentrations of the3-4H7 antibodies (0, 1 and 10 μg/ml). The vertical axis represents thesecretion amount of G-CSF (pg/10⁴ cells).

FIG. 9: Epitope mapping of the 3-4H7 antibody using the partial peptidesfrom MMRP19. The horizontal axis represents the type of peptidefragments. The vertical axis represents adsorbancy at 405 nm.

FIG. 10: Reactivity of the 3-4H7 antibody to COS7 cells. This figureshows the flow cytometric analysis of the expression in a monkey-derivedcell line COS7 of a human counterpart of the MMR19 gene. The horizontalaxis represents fluorescence intensity, and the vertical axis representsthe cell number. C represents non-transformed control COS7 cells, and HMrepresents the COS7 cells, in which a human counterpart of the MMRP19protein is expressed. Both the cells are stained with the 3-4H7antibody.

FIG. 11: Excessive expression of MMRP19 in RAW264.7 cells. This figureshows the flow cytometric analysis of which the expression in RAW264.7cells (an non-transformed cell line) or in RAW264.7 cells where theMMR19 gene is excessively expressed (OE-RAW264.7; transformed cells) ofa human counterpart of the MMR19 gene. The horizontal axis representsfluorescence intensity, and the vertical axis represents the cellnumber.

FIG. 12: Induction of G-CSF gene expression in OE-RAW264.7 cells wherean excessive amount of the MMRP19 protein is expressed. Each numberrepresents time after stimulation, and the upper case represents theband of G-CSF amplified by PCR whereas the lower case represents theband of a control G3PDH amplified by PCR.

FIG. 13: Increase in the secreted amount of G-CSF upon stimulation withthe 3-4H7 antibody to the cells where the MMRP19 is excessivelyexpressed. The secreted amount of G-CSF upon stimulation with the 3-4H7antibody to the RAW264.7 cells or OE-RAW264.7 cells is detected byELISA. The horizontal axis represents the time course, and the verticalaxis represents absorbance at 405 nm (the secreted amount of G-CSF).

FIG. 14: The mouse G-CSF mRNA induction of the 3-4H7 antibody againstthe peritoneal macrophage cells and the bone marrow cells. Peritonealmacrophage cells (0.5×10⁶) or bone marrow cells (1.5×10⁶) are left inculture plates for 18 hours. The 3-4H7-antibody or LPS are then addedthere and further cultured for 6 hours. After the 6 hours culture, RNAis extracted using an RNeasy kit, and the amount of the mouse G-CSF mRNAis measured by quantitative RT-PCR (PRISM 7700). Moreover, the amount ofGAPDH mRNA is measured as an internal standard so as to correct theamount of RNA.

FIG. 15: The mouse G-CSF mRNA induction of the 3-4H7 antibody againstthe mouse liver-derived Kupffer cells. Kupffer cells are prepared fromthe mouse liver, and 3-4H7 antibodies (10, 30 μg/ml) or LPS (1 μg/ml)are added there. After 1, 3 and 6 hours, the Kupffer cells are collectedand RNA is extracted using an RNeasy kit. The amount of the mouse G-CSFmRNA is measured by quantitative RT-PCR (PRISM 7700). Moreover, theamount of GAPDH mRNA is measured as an internal standard so as tocorrect the amount of RNA.

FIG. 16: The mouse G-CSF mRNA induction of the 3-4H7 antibody inmacrophage cells. Mouse adherent peripheral monocytes are cultured in amedium containing a macrophage colony-stimulating factor (30 ng/ml) for4 days so that the cells are differ ntiated into macrophage cells. 3-4H7antibodies (10, 30 μg/ml) or LPS (1 μg/ml) are added to the macrophagecells. After 15 minutes and 1 hour, the macrophage cells are collectedand then the m-RNA is extracted using an RNeasy kit. The amount of themouse G-CSF RMA is measured by quantitative RT-PCR (PRISM 7700).Moreover, the amount of GAPDH mRNA is measured as an internal standardso as to correct the amount of RNA.

FIG. 17: The mouse G-CSF mRNA induction of the peritoneal macrophagecells by the administration of the 3-4H7 antibody. The 3-4H7 antibody(4.2 mg/kg) is intraperitoneally administered, and after 3, 6 and 9hours, peritoneal macrophage cells are collected and followed by theextraction of RNA using an RNeasy kit. The amount of the mouse G-CSFmRNA is measured by quantitative RT-PCR (PRISM 7700). Moreover, theamount of GAPDH mRNA is measured as an internal standard so as tocorrect the amount of RNA.

FIG. 18: The effect of the 3-4H7 antibody on cyclophosphamide inducedmyelosuppressed mice. Cyclophosphamide (250 mg/kg) is intraperitoneallyadministered to the mice. On the third day after the initiation of theadministration of cyclophosphamide, a solvent, the 3-4H7 antibody (4.2mg/kg) is intraperitoneally administered for 3 days. As a positivecontrol, rhG-CSF (10 μg/kg) is subcutaneously administered (N=6). Sevendays after the initiation of the administration of cyclophosphamide, theblood is collected from the orbital venous plexus of the mice, and thenthe number of each of peripheral leukocytes, erythrocytes andthrombocytes is counted using Sysmex F-800. The smear of the same bloodsample is subjected to stain with May-Grünwald-Giemsa solution to countthe percentage of neutrophils. Statistical analysis is performed usingthe one way analysis of variance (one way ANOVA), LSD test is carriedout.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to specify an antigenrecognized by a G-CSF inducing antibody. It is another object of thepresent invention to clone and identify a gene encoding the antigenrecognized by the G-CSF inducing antibody.

Further, it is another object of the present invention to analyze indetail such an antigen protein and to clarify that the protein isassociated with the induction of G-CSF gene expression and/or G-CSFprotein secretion. Furthermore, it is another object of the presentinvention to provide a series of procedures for screening a compoundwhich changes the generation of G-CSF gene expression and/or G-CSFprotein induction, using the above protein.

During intensive studies directed towards the above objects, the presentinventors have carried out the immunoscreening of a cDNA library derivedfrom macrophage cells, using a monoclonal antibody with ability toinduce G-CSF as a probe, As a result, the present inventors havesucceeded in the isolation of a positive clone and they have determinedits nucleotide sequence, thereby providing the present invention.Moreover, the present inventors have also determined the nucleotidesequence of a human counterpart antigen gene.

That is to say, the present invention provides a gene encoding a protein(a), (b) or (c) described as follows; (a) a protein having an amino acidsequence shown in SEQ ID NO: 2; (b) a protein having an amino acidsequence comprising a deletion, substitution, addition or insertion ofone or several amino acids with respect to the amino acid sequence shownin SEQ ID NO: 2, and having a binding ability for an antibody capable ofinducing and secreting a G-CSF or a fragment thereof; or (c) a proteinhaving at least 50% or more homology with the amino acid sequence shownin SEQ ID NO: 2, and having a binding ability for an antibody capable ofinducing and secreting a G-CSF or a fragment thereof.

Moreover, the present invention provides a gene encoding a protein (a),(b) or (c) described as follows: (a) a protein having an amino acidsequence shown in SEQ ID NO: 4; (b) a protein having an amino acidsequence comprising a deletion, substitution, addition or insertion ofone or several amino acids with respect to the amino acid sequence shownin SEQ ID NO: 4, and having a binding ability for an antibody capable ofinducing and secreting a G-CSF or a fragment thereof; or (c) a proteinhaving at least 50% or more homology with the amino acid sequence shownin SEQ ID NO: 4, and having a binding ability for an antibody capable ofinducing and secreting a G-CSF or a fragment thereof.

Further, the present invention provides a gene having a nucleotidesequence (a), (b) or (c) described as follows: (a) a nucleotide sequenceshown in SEQ ID NO: 1; (b) a nucleotide sequence comprising a deletion,substitution, addition or insertion of one or several nucleotides withrespect to the nucleotide sequence shown in SEQ ID NO: 1, and encoding aprotein having a binding ability for an antibody capable of inducing andsecreting a G-CSF or a fragment thereof; or (c) a nucleotide sequencewhich hybridizes with DNA having the nucleotide sequence shown in SEQ IDNO: 1 under stringent conditions, and encoding a protein having abinding ability for an antibody capable of inducing and secreting aG-CSF or a fragment thereof.

Furthermore, the present invention provides a gene having a nucleotidesequence (a). (b) or (c) described as follows: (a) a nucleotide sequenceshown in SEQ ID NO: 3; (b) a nucleotide sequence comprising a deletion,substitution, addition or insertion of one or several nucleotides withrespect to the nucleotide sequence shown in SEQ ID NO: 3, and encoding aprotein having a binding ability for an antibody capable of inducing andsecreting a G-CSF or a fragment thereof; or (c) a nucleotide sequencewhich hybridizes with DNA having the nucleotide sequence shown in SEQ IDNO; 3 under stringent conditions and encoding a protein having a bindingability for an antibody capable of inducing and secreting a G-CSF or afragment thereof.

In the above description, the antibody capable of inducing and secretinga G-CSF is, for example, a monoclonal antibody produced from hybridomacells deposited under accession No. FERM BP-6103.

The gene of the present invention is, for example, a gene derived frommouse or human.

The present invention provides a DNA fragment comprising any of thefollowing nucleotide sequences: (1) a nucleotide sequence consisting ofnucleotides 519 to 736, nucleotides 666 to 689, nucleotides 381 to 403,or nucleotides 709 to 727 with respect to the nucleotide sequence shownin SEQ ID NO: 1: (2) a nucleotide sequence comprising a deletion,substitution, addition or insertion of one or several nucleotides withrespect to the nucleotide sequence of (1) above; and (3) a nucleotidesequence having at least 80% homology with any one of the nucleotidesequences of (1) above.

Moreover, the present invention provides a gene having any one of thefollowing nucleotide sequences: (1) a nucleotide sequence consisting ofnucleotides 519 to 736, nucleotides 666 to 689. nucleotides 381 to 403,or nucleotides 709 to 727 with respect to the nucleotide sequence shownin SEQ ID NO: 1: (2) a nucleotide sequence comprising a deletion,substitution, addition or insertion of one or several nucleotides withrespect to the nucleotide sequence of (1) above: and (3) a nucleotidesequence having homology of at least 80% with any one of the nucleotidesequences of (1) above, and encoding a protein having a binding abilityfor an antibody capable of inducing and secreting a G-CSF or a fragmentthereof.

Further, the present invention provides a protein (a), (b), (c) or (d)described as follows: (a) a protein having an amino acid sequence shownin SEQ ID NO: 2; (b) a protein having an amino acid sequence comprisinga deletion, substitution, addition or insertion of one or several aminoacids with respect to the amino acid sequence shown in SEQ ID NO: 2, andhaving a binding ability for an antibody capable of inducing andsecreting a G-CSF or a fragment thereof: (c) a protein having at least50% or more homology with the amino acid sequence shown in SEQ ID NO: 2,and having a binding ability for an antibody capable of inducing andsecreting a G-CSF or a fragment thereof: or (d) a protein encoded by DNAwhich hybridizes with DNA having the nucleotide sequence shown in SEQ IDNO: 1 under stringent conditions, and having a binding ability for anantibody capable of inducing and secreting a G-CSF or a fragmentthereof.

Furthermore, the present invention provides a protein (a), (b), (c) or(d) described as follows: (a) a protein having an amino acid sequenceshown in SEQ ID NO: 4; (b) a protein having an amino acid sequencecomprising a deletion, substitution, addition or insertion of one orseveral amino acids with respect to the amino acid sequence shown in SEQID NO: 4, and having a binding ability for an antibody capable ofinducing and secreting a G-CSF or a fragment thereof: (c) a proteinhaving at least 50% or more homology with the amino acid sequence shownin SEQ ID NO: 4, and having a binding ability for an antibody capable ofinducing and secreting a G-CSF or a fragment thereof; or (d) a proteinencoded by DNA which hybridizes with DNA having the nucleotide sequenceshown in SEQ ID NO: 3 under stringent conditions, and having a bindingability for an antibody capable of inducing and secreting a G-CSF or afragment thereof.

In the above description, the antibody capable of inducing and secretinga G-CSF is, for example, a monoclonal antibody produced from hybridomacells deposited under accession No. FERM BP-6103.

The protein of the present invention is derived preferably from mammaland particularly preferably from mouse or human.

The present invention provides a protein having any one of the followingamino acid sequences: (1) an amino acid sequence consisting of aminoacids 1 to 91, amino acids 50 to 146, amino acids 1 to 78, amino acids200 to 241, amino acids 172 to 241, amino acids 103 to 150, or aminoacids 169 to 241 with respect to the amino acid sequence shown in SEQ IDNO: 2; (2) an amino acid sequence comprising a deletion, substitution,addition or insertion of one or several amino acids with respect to theamino acid sequence of (1) above; and (3) an amino acid sequence havingat least 70% homology with any one of the amino acid sequences of (1)above.

Moreover, the present invention provides a protein having any one of thefollowing amino acid sequences; (1) an amino acid sequence consisting ofamino acids 1 to 91, amino acids 50 to 146, amino acids 1 to 78, aminoacids 200 to 241, amino acids 172 to 241, amino acids 103 to 150, oramino acids 169 to 241 with respect to the amino acid sequence shown inSEQ ID NO: 2: (2) an amino acid sequence comprising a deletion,substitution, addition or insertion of one or several amino acids withrespect to the amino acid sequence of (1) above: and (3) an amino acidsequence having at least 70% homology with any one of the amino acidsequences of (1) above, and having a binding ability for an antibodycapable of inducing and secreting a G-CSF or a fragment thereof.

Further, the present invention provides an antibody against theabovedescribed protein of the present invention or a fragment thereof.The antibody is preferably a monoclonal antibody, and particularlypreferably a human-type monoclonal antibody or human monoclonalantibody.

Furthermore, the present invention provides a recombinant vectorcomprising the gene of the present invention or a DNA fragment thereof.

Furthermore, the present invention provides a transformant comprising arecombinant vector comprising the gene of the present invention or a DNAfragment thereof.

Furthermore, the present invention provides a novel receptor or aportion thereof (the protein of the present invention) which is capableof inducing or promoting the secretion of a G-CSF.

Furthermore, the present invention provides a method of screening auseful substance (e.g. an agonist or antagonist of the protein of thepresent invention) by using the protein of the present invention, asubstance obtained by the screening method and a useful substancecapable of binding to a receptor (e.g. an agonist or antagonist of thereceptor.)

Furthermore, the present invention provides a pharmaceutical compositioncomprising the gene, DNA fragment, protein (including a fragment of theprotein) antibody (including a fragment thereof), receptor, or substance(including a low molecular compound) of the present invention (thepharmaceutical composition particularly used for diagnosis, preventionor treatment of infectious diseases, neutropenia, or cytopenia regardingthe reduction of the number of erythrocytes, leukocytes orthrombocytes); and a treatment method of using the pharmaceuticalcomposition.

The embodiments and methods for carrying out the present invention willbe explained in detail below.

Prior to the present invention, the present inventors obtained anantibody by the immunization of a macrophage itself, and they succeededin isolating an antibody which induces G-CSF from the obtained antibody(Japanese Patent Application No. 9-266591; the disclosure of thisapplication is incorporated herein by reference in its entirety). Thegene of the present invention was isolated by screening a cDNA libraryderived from the mouse macrophage, using this antibody as a probe. Theprotein encoded by the gene of the present invention is characterized inthat it has a binding ability to an antibody capable of inducing andsecreting a G-CSF or a fragment thereof.

<Antibody which Induces or Promotes Secretion of G-CSF, or FragmentThereof>

First, with regard to “an antibody which induces or promotes secretionof G-CSF or a fragment thereof” of the present specification(hereinafter referred to also as “antibody used in the presentinvention”), a method of obtaining the same or the like will beexplained.

First, the present inventors administered cells of the mouse macrophagecell line as an antigen to MRL/lpr mice (autoimmune-disease mouse) andisolated a monoclonal antibody. Then they treated the obtainedmoloclonal antibodies to the immunized cell, a mouse macrophage cellline, in order to study the effect of the antibody to the immunizedcell. As a result, they found that one of the obtained antibodies has aproperty to concentration-dependently induce G-CSF from the immunizedcell line, the mouse macrophage cell line (a hybridoma producing thisantibody is deposited under international accession No. FERM BP-6103).

The term “monoclonal antibody” is used in the present specification tomean a monoclonal antibody having reactivity with a macrophage cellline, and specifically, it is a monoclonal antibody having an activitywhich promotes the generation of G-CSF.

The antibody used in the present invention has a property tosubstantially bind to a macrophage cell line. The antibody used in thepresent invention includes both a polyclonal antibody and a monoclonalantibody, which have the abovedescribed property. Moreover, the“monoclonal antibody” includes monoclonal antibodies belonging to anyimmunoglobulin class such as IgG, IgM, IgA, IgD and IgE, and it ispreferably an IgG or IgM immunoglobulin class monoclonal antibody.

The macrophage cell line can be prepared from spontaneous leukemiccells, or it can be also prepared by cellular transformation by leukemicviruses.

The antibody used in the present invention can be obtained according tothe conventional methods (e.g. a method described in the publication“Zoku Seikagaku Jikken Koza 5, Meneki Seikagaku Kenkyu Ho, edited by TheJapanese Biochemical Society, published by Tokyo Kagaku Dojin Co. Ltd.).

The monoclonal antibody used in the present invention can be producedfrom a hybridoma (a hybrid cell) which is produced by what is calledcell fusion. That is to say, a hybridoma is obtained from an antibodygenerating cell and a myeloma cell, the obtained hybridoma is cloned,and using all or a part of macrophage cell lines as an antigen, a clonegenerating an antibody showing a specific affinity toward the antigen isselected as the monoclonal antibody used in the present invention. Toproduce the monoclonal antibody, previously known methods can be appliedwith the exception that all or a part of macrophage cell lines is usedas an immunogen.

The immunogen is obtained by, for example, the direct use of amacrophage cell line, or it is prepared by mixing the whole or a portionof the membrane fraction or soluble extract of a macrophage cell linewith e.g. a complete Freund's adjuvant as necessary. Examples of animalsused as the target of immunization include mammals such as mouse, rat,guinea pig, hamster or rabbit, preferably mouse or rat, and particularlypreferably mouse. Immunization is carried out by injection one toseveral times into the subcutis, muscle, vein, footpad or peritonealcavity of the above described mammals.

Generally, the subsequent immunization is carried out once to four timesabout every one to two weeks after the initial immunization, and thenabout one to four weeks later, the final immunization is carried out.About three to five days after the final immunization, antibodyproducing cells are collected from the immunized aminal.

The monoclonal antibody used in the present invention includes amonoclonal antibody (3-4H7 antibody) produced from hybridoma cellsdeposited under “international accession No. FERN BP-6103”, a fragmentthereof, and an antibody having substantially the same properties as theabove antibody. The “3-4H7 antibody” has an ability to generate G-CSFfrom the cells.

The hybridoma producing the monoclonal antibody used in the presentinvention can be prepared by known methods. Such known methods include,for example, Köhler and Milstein's method (Nature, Vol. 256, pp.495-497, 1975) and other modified methods equivalent thereto, for apreparation of a hydridoma which secretes a monoclonal antibody. Thatis, a monoclonal antibody can be prepared by culturing the hybrid cells(hybridomas), which are obtained by fusion between an antibody producingcells in the spleen, lymph node, bone marrow or tonsilla, preferably thespleen from the animal immunized as above, and myeloma cells (myelomas)obtained from preferably the same kind of mammal such as mouse, rat,guinea pig, hamster, rabbit or human, and more preferably from mouse,rat or human. Cultures can be carried out in vitro, or cells are grownin vivo, for example, in the ascites of mammals such as mouse, rat,guinea pig, hamster or rabbit, preferably of mouse or rat, and morepreferably of mouse, and the antibody can be obtained from the culturesupernatant or the ascites of the mammals.

Examples of myeloma cells used in a cell fusion include myelomas“P3/X63-AG8”, “P3/NS1/1-Ag4-1”, “P3/X63-Ag8.U1”, “SP2/0-Ag14”, “PAI”,“FO” and “BW5147”, which are derived from mouse, a myeloma“21ORCY3-Ag1.2.3”, which is derived from rat, and myelomas “U-266AR1”,“GM1500-6TG-A1-2”, “UC729-6”, “CEM-AGR”, “D1R11” and “CEM-T15”, whichare derived from human.

Screening of a hybrid cell clone producing the monoclonal antibody usedin the present invention can be carried out by culturing a hybrid cell,for example, in a microtiter plate, and determining the reactivity of anantigen of the culture supernatant in the well where proliferation ofthe cells is observed, for example, by flow cytometry or by enzymatictechniques such as RIA or ELISA.

Examples of a basal medium include low calcium-medium such as Ham's F12medium, MCDB153 medium or low calcium-MEM medium: high calcium-mediumsuch as MCDB104 medium, MEM medium, D-MEM medium, RPM11640 medium,ASF104 medium or RD medium; and others. Depending on purposes, to theabove basal medium, sera, hormones, cytokines, and/or various inorganicor organic substances can be added. Isolation and purification of amonoclonal antibody can be carried out by subjecting the above describedculture supernatant or ascites to collect antibodies by ammonium sulfateprecipitation, euglobulin precipitation, caproic acid method, caprylicacid method, ion-exchange chromatography (DEAE, DE52 or the like),affinity column chromatography, which uses an anti-immunoglobulin columnor a protein A or G column, hydrophobic chromatography, and others.

The monoclonal antibody used in the present invention can be obtained byany of the method, which are not limited to the above productionmethods. Generally, a “monoclonal antibody” has a sugar chain, thestructure of which is different depending on the types of a mammal to beimmunized. However, the “monoclonal antibody” used in the presentinvention is not limited by the structural difference of the sugarchain, but it includes monoclonal antibodies derived from any type ofmammals. The “monoclonal antibody” used in the present inventionincludes: a monoclonal antibody produced by phage display; a human-typemonoclonal antibody obtained by, for example, using a transgenic mouse,which is produced by introducing a human immunoglobulin gene throughgene engineering so as to generate a human-type antibody; a chimericmonoclonal antibody obtained by substituting the constant region (Fcregion) of a monoclonal antibody derived from a certain mammal with theFe region of a human monoclonal antibody by genetic engineeringtechniques: and a humanized monoclonal antibody obtained by substitutingthe entire region other than complementarity-determining regions (CDRs),which have complementarily and directly binding to an antigen, with acorresponding region of human monoclonal antibody.

In the present invention, the “fragment of an antibody” may also beused. The term “the fragment of an antibody” is used herein to mean anantibody fragment comprising at least one variable region, and the termis the same definition as a “portion of an antibody” described inJapanese Patent Application No. 9-266591. Specifically, the antibodyfragment refers to Fv, F(ab′)2, Fab′ or Fab. Each of the terms “F(ab′)2”and “Fab′” are used herein to mean an antibody fragment obtained bytreating immunoglobulin (monoclonal antibody) with protease such aspepsin or papain and digesting before or after the disulfide bondsbetween two H chains in a hinge region. For example, when IgG is treatedwith papain, it is cleaved upstream of the disulfide bond between thetwo H chains in the hinge region, thereby producing two homologousantibody fragments in which an L chain fragment consisting of VL (an Lchain variable region) and CL (an L chain constant region) and an Hchain fragment consisting of VH (an H chain variable region) and CHγ1 (aγ1 region in an H chain constant region) are bound in a C-terminalregion by a disulfide bond. Each of the two homologous antibodyfragments is called Fab′. When IgG is treated with pepsin, it is cleaveddownstream of the disulfide bonds between the two H chains in the hingeregion, thereby producing an antibody fragment which is slightly largerthan the above described two Fab's which are bound with each other inthe hinge region. This antibody fragment is called F(ab′)2.

The protein encoded by the gene of the present invention ischaracterized in that it has a binding ability to the antibody capableof inducing and secreting a G-CSF or a fragment thereof, as described indetail above. The term “affinity” is used in the present specificationto mean an ordinary affinity between a protein and an antibody, and itcan be determined by commonly used immunological analyses (e.g.immunoprecipitation method, ELISA, immunoblotting, etc.).

<Gene of the Present Invention>

The present invention provides a gene encoding a protein having an aminoacid sequence shown in SEQ ID NO: 1 or a protein homologous to the aboveprotein. The present invention further provides a gene having anucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequencehomologous to the above nucleotide sequence.

The type of the gene of the present invention is not particular limited.Any of native DNA, recombinant DNA and chemically synthesized DNA may beused, and further, a genomic DNA clone and a cDNA clone may also beused.

The gene of the present invention typically has a nucleotide sequenceshown in SEQ ID NO: 1. This is the nucleotide sequence of a clone(MMR19) obtained in the examples as described later, which are providedfor illustrative purposes only. It is well known to a person skilled inthe art that native genes include a few mutants caused by the differenceof the species of an organism which produces the genes, a few mutantscaused by difference in the ecosystem, or by the presence of a verysimilar isozyme. Accordingly, the gene of the present invention is notlimited to the gene having the nucleotide sequence shown in SEQ ID NO:1, but it includes all the genes encoding the proteins having featuresdescribed in the present specification.

The description “comprising a deletion, substitution, addition orinsertion of one or several nucleotides” is used in the presentspecification to mean that a certain number of nucleotides aresubstituted by known techniques such as a site-directed mutagenesis, orthese are spontaneously substituted. The number of nucleotidessubstituted is, for example, 10 or less, and preferably 3 to 5 or less.

If the amino acid sequence of the protein of the present invention and aDNA sequence encoding the same are disclosed in the presentspecification, using these sequences or portions thereof, a geneencoding a protein having similar physiological activity as theinventive protein can easily be isolated from other organisms by basicgene engineering techniques such as hybridization and PCR. In such acase, the thus obtained gene is also included in the scope of thepresent invention.

Hybridization conditions for screening homologous genes are notparticularly limited. A person skilled in the art can appropriatelyselect hybridization conditions, considering the level of homologybetween a homologous gene of interest and a probe. However, in general,stringent conditions are preferable, and an example includeshybridization conditions of 6×SSC [0.9 M NaCl, 0.09 M sodium citrate (pH7.0)], 5× Denhardt's solution [1 g of ficoll. 1 g ofpolyvinylpyrrolidone and 1 g of BSA in 1,000 mL], 0.5% SDS, and 25° C.to 68° C. (for example, 37° C., 42° C. or 68° C.), or hybridizationconditions of 0% to 50% formamide, 6×SSC, 0.5% SDS, and 25° C. to 68° C.(for example, 37° C., 42° C. or 68° C.). It is well known to thoseskilled in the art that DNA having a nucleotide sequence with more thana certain level of homology can be cloned by appropriately settinghybridization conditions such as formamide concentration, Denhardt'ssolution concentration, salt concentration and temperature. All the thuscloned homologous genes are also included in the scope of the presentinvention.

Homologous genes cloned by the above described hybridization have atleast 70% or more, preferably 80% or more, more preferably 90% or more,particularly preferably 95% or more, and most preferably 98% or morehomology with the nucleotide sequence of SEQ ID NO: 1.

<Protein of the Present Invention>

The present invention provides a protein having an amino acid sequenceshown in SEQ ID NO: 1 or a protein having homology with the protein.

The protein having the amino acid sequence shown in SEQ ID NO: 1 of thepresent invention can be obtained by integrating a gene encoding theprotein into a suitable expression vector and then transfecting thevector into a suitable host so that a recombinant protein is allowed toexpress therein. However, the origin or production method of the proteinof the present invention is not limited, as long as the protein hasfeatures described in the present specification. Accordingly, any of anative protein, a protein expressed from recombinant DNA by geneticengineering, and chemically synthesized protein may be used.

The protein of the present invention typically has an amino acidsequence consisting of 241 amino acids shown in SEQ ID NO: 1. However,it is well known that native proteins include mutant proteins comprisingone to several amino acid mutants, which are caused by the differenc ofthe species of an organism which produces the proteins, gene mutationcaused by difference in the ecosystem, or the presence of a very similarisozyme. The term “amino acid mutants” are used herein to mean asubstitution, deletion, insertion and/or addition of one or more aminoacids. When an assumption is made from the nucleotide sequence of thecloned gene, the protein of the present invention has the amino acidsequence shown in SEQ ID NO: 1. However, the protein of the presentinvention is not limited to a protein having that amino acid sequence,but it is intended to include all homologous proteins, as long as theyhave features described in the present specification. The level ofhomology is at least 50% or more, preferably 60% or more, morepreferably 70% or more, further preferably 80% or more, further morepreferably 90% or more, particularly preferably 95% or more, and mostpreferably 98% or more.

Generally, when amino acids having similar features are substituted(e.g. substitution of hydrophobic amino acids, substitution ofhydrophilic amino acids, substitution of acidic amino acids, orsubstitution of basic amino acids), in many cases, the obtained mutantprotein has the same features as those of the original protein. A methodof producing a recombinant protein by genetic engineering having certaindesired mutations is well known to a person skilled in the art, and sucha mutant protein is also included in the scope of the present invention.

When the description “a deletion, substitution, addition or insertion ofone or several amino acids” is used for an amino acid sequence in thepresent specification, it means that a certain number of amino acids aresubstituted by known techniques such as a site-directed mutagenesis, orthese are spontaneously substituted. The number of amino acidssubstituted is, for example, 10 or less, and preferably 3 to 5 or less.The term “homology” regarding amino acid sequences is used herein tomean the level of the correspondence between amino acid residues whichconstitute amino acid sequences to be compared. This time, the presenceof gaps and the properties of amino acids are taken into consideration(Wilbur, Proc. Natl. Acad. Sci. USA 80: 726-730 (1983), etc.) In orderto calculate homology, commercially available software such as BLAST(Altschul: J. Mol. Biol. 215: 403-410 (1990)), FASTA (Peasron: Methodsin Enzymology 183: 63-69 (1990)), Genetyx-Mac (Software Development Co.,Ltd.) and others can be used.

In examples described later in the present specification, cloning ofcDNA derived from the mouse macrophage is shown as an example of thepresent invention. Isolation of a gene encoding a protein having thesame physiological activity as the inventive protein from other originsby genetic engineering such as hybridization and PCR, using the aminoacid sequence of the protein disclosed in the present specification thesequence of a gene encoding the protein (d rived from mouse) or aportion thereof, is within the common technical knowledge of a personskilled in the art, and a protein encoded by the thus isolated gene isalso included in the scope of the present invention.

<Human-type Gene and Protein>

An example of a method for obtaining a human-derived homolog withrespect to the gene and protein of the present invention is as follows.

Total RNA is extracted from human macrophage cell lines (THP-1, U937,HL-60) by guanidium thiocyanate/phenol chloroform single step extraction(LaboManual Gene Engineering, 3^(rd) edition, pages 83 and 84, 1996),and the total RNA is then purified using an oligo(dT) cellulose columnto obtain poly A⁺RNA. Using reverse transcriptase (MMLV-RTase) and DNApolymerase I, double stranded cDNA is synthesized. A cDNA library isconstructed from the obtained double stranded cDNA according toGubler-Hoffmann method (Gubler, U. and Hoffmann. B., J.: Gene, 25:263-269, 1983), using a λZAPII phage vector. Using a DNA sequence as aprobe, which is amplified using the cDNA library of human macrophagecells as a template and using primers capable of amplifying a sequencein the region which has high homology with human (e.g. a regioncorresponding to nucleotides 172 to 241 of the nucleotide sequence shownin SEQ ID NO: 1, which has 91% homology with human) in the nucleotidesequence (SEQ ID NO: 1) of mouse cDNA (MMR19 clone) disclosed in thepresent specification: or directly using such a region (e.g. a regioncorresponding to nucleotides 172 to 241 of the nucleotide sequence shownin SEQ ID NO: 1) as a probe, the EDNA library of human macrophage cellsis screened to isolate cDNA encoding the full length amino acid sequenceof a protein of interest. Thereafter, according to Primer Walkingmethod, the nucleotide sequence of the obtained cDNA is analyzed. Afterconfirming that the cDNA encodes the full length of the protein ofinterest, the cDNA is introduced into baculovirus so as to allow it toexpress itself as a protein. Thereafter, the protein is purified usingan affinity column so that a human-type homologue protein can beobtained.

As stated above, the present invention relates to a gene having thenucleotide sequence shown in SEQ ID NO: 1, a protein having the aminoacid sequence shown in SEQ ID NO: 2, and genes and proteins havinghomology with the above gene and protein. A search was made regardingwhether or not a nucleotide sequence and an amino acid sequence havinghomology with the nucleotide sequence shown in SEQ ID NO: 1 and theamino acid sequence shown in SEQ ID NO: 2, which are provided by thepresent invention are present in other organisms. As a result, it wasconfirmed that a gene having high homology with the gene of the presentInvention is present in human EST (expressed sequence tag) (refer toExample 3 described later). Accordingly, it is clear that ahuman-derived homologue gene can also be isolated by screening ahuman-derived gene library (cDNA library, etc.), using, as a probe,human-derived EST having high homology with the nucleotide sequence ofthe present invention.

As stated above, the search through the database clarified that aportion of the nucleotide sequence shown in SEQ ID NO: 1 of the presentinvention (that is, a DNA fragment) is also present in human with highhomology. Such a DNA fragment, as stated above, is useful as a probewhen a human-derived homologue gene is screened, and it forms an aspectof the present invention. Examples of such a DNA fragment include a DNAfragment having any one of a nucleotide sequence consisting ofnucleotides 519 to 736, a nucleotide sequence consisting of nucleotides666 to 689, a nucleotide sequence consisting of nucleotides 381 to 403,and a nucleotide sequence consisting of nucleotides 709 to 727 withrespect to the nucleotide sequence shown in SEQ ID NO: 1. Further, a DNAfragment having a nucleotide sequence comprising a deletion,substitution, addition or insertion of one or several nucleotides withrespect to any one of the above nucleotide sequences; or a DNA fragmenthaving a nucleotide sequence having at least 80%, preferably 85% ormore, more preferably 90% or more, further preferably 95% or more, andmost preferably 98% or more homology with any one of the abovenucleotide sequences, is also within the scope of the present invention.

Moreover, the search through the database also clarified that a portionof the amino acid sequence shown in SEQ ID NO: 2 of the presentinvention is present in human with high homology. As with the protein ofthe present invention, a protein fragment consisting of a portion of theprotein of the present invention is useful as a reagent for analyzing orisolating an antibody capable of inducing and secreting G-CSF, and aswith the protein of the present invention, the protein fragment islikely to be used as a pharmaceutical. Accordingly, the protein fragmentforms an aspect of the present invention.

Examples of such a protein include a protein having any one of an aminoacid sequence consisting of amino acids 1 to 91, an amino acid sequenceconsisting of amino acids 50 to 146, an amino acid sequence consistingof amino acids 1 to 78, an amino acid sequence consisting of amino acids200 to 241, an amino acid sequence consisting of amino acids 172 to 241,an amino acid sequence consisting of amino acids 103 to 150, and anamino acid sequence consisting of amino acids 169 to 241 with respect tothe amino acid sequence shown in SEQ ID NO: 2. Further, a protein havingan amino acid sequence comprising a deletion, substitution, addition orinsertion of one or several amino acids with respect to any one of theabove amino acid sequences; or a protein having an amino acid sequencehaving at least 70%, preferably 80% or more, more preferably 85% ormore, further preferably 90% or more, and further more preferably 95% ormore, and most preferably 98% or more homology with any one of the abovenucleotide sequences, is also included in the scope of the presentinvention.

The present inventors have determined the nucleotide sequence of ahuman-type antigen gene according to a method similar to the methodsdescribed above (refer to Example 5 described later). Accordingly, thepresent invention provides a gene having a nucleotide sequence shown inSEQ ID NO: 3 or nucleotide sequence having homology therewith. Moreover,the present invention provides a protein having an amino acid sequenceshown in SEQ ID NO: 4 or protein having homology therewith. As describedin the section “Gene of the present invention” or “Protein of thepresent invention” of the present specification, the meaning of homologyused herein, that is, the scope of the present invention, is not limitedto the gene having the nucleotide sequence shown in SEQ ID NO: 3 or theprotein having the amino acid sequence shown in SEQ ID NO: 4.

<Antibody of the Present Invention>

The present invention provides an antibody against the above describedprotein of the present invention (hereinafter referred to also as “themonoclonal antibody of the present invention.”) Embodiments and methodsof obtaining the antibody of the present invention will be explained indetail below.

The antibody of the present invention may be a polyclonal antibody ormonoclonal antibody. In the case of a monoclonal antibody, it may be achimeric antibody, and a mouse/human chimeric antibody is particularlypreferable. The “monoclonal antibody” includes monoclonal antibodiesbelonging to any immunoglobulin class such as IgG, IgM, IgA, IgD andIgE, and it is preferably an IgG or IgM immunoglobulin class monoclonalantibody.

The protein of the present invention, which is used as an antigen, canbe obtained by integrating a gene encoding the protein into a suitableexpression vector and then transfecting the vector into a suitable hostso that a recombinant protein is allowed to be expressed therein.Examples of an immunogen used include a macrophage cell line itself andthe membrane fraction of the macrophage cell line.

The antibody of the present invention such as a polyclonal antibody(antiserum) or monoclonal antibody can be obtained by conventionalmethods (e.g. a method described in the publication “Zoku SeikagakuJikken Koza 5, Meneki Seikagaku Kenkyu Ho, edited by The JapaneseBiochemical Society, published by Tokyo Kagaku Dojin Co. Ltd.)

That is to say, for example, a mammal that is preferably mouse, rat,hamster, guinea pig, rabbit, dog, cat, pig, goat, horse or cow, and morepreferably mouse, rat, hamster, guinea pig or rabbit, is immunized withan antigen together with Freund's adjuvant if necessary. A polyclonalantibody can be obtained from serum obtained from the thus immunizedmammal. A monoclonal antibody can be produced by preparing a hybridomafrom an antibody producing cell obtained from the immunized mammal and amyeloma cell (myeloma) having no ability to produce an autoantibody,cloning the hybridoma, and selecting a clone producing a monoclonalantibody which has specific affinity toward the antigen used forimmunizing the mammal.

Specifically, the monoclonal antibody can be produced as follows. Thatis, the protein of the present invention or a cell expressing theprotein of the present invention or the like are used as an immunogen,mouse, rat, hamster, guinea pig or rabbit, or preferably mouse, rat orhamster (these animals also include transgenic animals such as a humanantibody producing transgenic mouse, which are produced to generateantibodies derived from other animals) is immunized with the aboveimmunogen by injecting the immunogen together with Freund's adjuvant asnecessary into the subcutis, muscle, vein, footpad or peritoneal cavityof the above mammal one to several times, or by transplanting it thereinfor immunization. Generally, after the initial immunization, thesubsequent immunizations are carried out one to four times every one tofourteen days, and about one to five days after the final immunization,an antibody producing cell is obtained from the immunized mammal.

The monoclonal antibody of the present invention can be produced from ahybridoma (hybrid cell), which is produced by what is called cellfusion.

Hybridoma producing the monoclonal antibody can be prepared by knownmethods. Examples of the known methods include Köhler and Milstein'smethod (Nature, Vol. 256, pp. 495-497. 1975) and other modified methodsequivalent thereto. That is, the monoclonal antibody of the presentinvention can be prepared by culturing the hybrid cell (hybridoma),which is obtained by fusion between an antibody producing cell containedin the spleen, lymph node, bone marrow or tonsilla, preferably thespleen obtained from the animal immunized as above, and a myeloma cell(myeloma) derived preferably from the same kind of mammal such as mouse,rat, guinea pig, hamster, rabbit or human, and more preferably frommouse, rat or human.

Examples of the myeloma cell (myeloma) used in cell fusion includemyelomas “P3/X63-AG8”, “P3/NS1/1-Ag4-1”, “P3/X63-Ag8.U1”, “SP2/0-Ag14”,“X63, 653”, “PAI”, “FO” and “BW5147”, which are derived from mouse, amyeloma “210RCY3-Ag1.2.3”, which is derived from rat, and myelomas“U-266AR1”, “GM1500-6TG-A1-2”. “UC729-6”, “CEM-AGR”, “D1R11” and“CEM-T15” which are derived from human.

Screening of a hybrid cell clone producing the monoclonal antibody ofthe present invention can be carried out by culturing a hybrid cell, forexample, in a microtiter plate, and determining the reactivity with anantigen of the culture supernatant in a well where proliferation of thecell is observed, for example, by flow cytometry, RIA, ELISA or thelike.

Production of the monoclonal antibody from hybridoma is carried out invitro, or in vivo, for example, in the ascites of mouse, rat, guineapig, hamster or rabbit, preferably of mouse or rat, and more preferablyof mouse, and the monoclonal antibody can be then isolated from theculture supernatant or the ascites of mammals. In the case of in vitroculture, hybridoma is proliferated, maintained and preserved, dependingon various conditions such as the properties of cells to be cultured,the purpose of the test and the culture method, and the culture can becarried out, using a known nutrient medium used to allow accumulation ofthe monoclonal antibody in the culture supernatant or any type ofnutrient medium prepared from known basal media.

Examples of the basal medium include a low-calcium medium such as Ham'sF12 medium, MCDB153 medium or a low-calcium MEM medium; high-calciummedium such as MCDB104 medium, MEM medium, D-MEM medium, RPMI1640medium, ASF104 medium or RD medium; and others. Depending on purposes,to the above basal medium, sera, hormones, cytokines, and/or variousinorganic or organic substances can be added. Isolation and purificationof the monoclonal antibody can be carried out by subjecting the abovedescribed culture supernatant or ascites to ammonium sulfateprecipitation, euglobulin precipitation, caproic acid method, caprylicacid method, ion exchange chromatography (DEAE, DE52 or the like),affinity column chromatography which uses an anti-immunoglobulin columnor a protein A or G column, hydrophobic chromatography, and others.

The “chimeric antibody” of the present invention is a monoclonalantibody produced by genetic engineering, and it specifically means achimeric monoclonal antibody such as a mouse/human chimeric monoclonalantibody, which is characterized by that its variable region is derivedfrom mouse immunoglobulin and its constant region is derived from humanimmunoglobulin. The constant region derived from a human immunoglobulinhas a specific amino acid sequence, depending on isotypes such as IgG,IgM, IgA, IgD and IgE. The recombinant chimeric monoclonal antibody ofthe present invention may comprise a constant region derived from ahuman immunoglobulin, which belongs to any of the isotypes. Preferably,it is a constant region derived from human IgG. The chimeric monoclonalantibody of the present invention can be produced, for example, asfollows: However, needless to say, the production method is not limitedto the following.

For example, a mouse/human chimeric monoclonal antibody can be produced,referring to Jikken Igaku (extra edition), Vol. 1.6, No. 10, 1988 andJapanese Patent Publication No. Hei3-73280, etc. That is to say, a C_(H)gene (a gene encoding an H chain constant region) obtained from DNAencoding human immunoglobulin is placed downstream of an active V_(H)gene (a rearranged VDJ gene encoding an H chain variable region)obtained from DNA encoding a mouse monoclonal antibody isolated fromhybridoma producing the monoclonal antibody so that the gene can beexpressed, and further, a C_(L) gene (a gene encoding an L chainconstant region) obtained from DNA encoding human immunoglobulin isplaced downstream of an active V_(L) gene (a rearranged VJ gene encodingan L chain variable region) obtained from DNA encoding a mousemonoclonal antibody isolated from the above hybridoma so that the genecan be expressed. Thereafter, these genes are then inserted into asingle or two separate expression vectors, a host cell is transformed bythe vector(s), and the obtained transformed cell is cultured so as toproduce the mouse/human chimeric monoclonal antibody.

Specifically, first, DNA is extracted from a mouse monoclonal antibodyproducing hybridoma by a conventional method, and the purified DNA isthen digested with suitable restriction enzymes (e.g. EcoRI, HindIII andothers). Thereafter, the digested DNA is subjected to electrophoresis(e.g. using 0.7% agarose gel) and Southern blotting. After subjectingthe gel to electrophoresis, the gel is stained with e.g. ethidiumbromide. A photograph of the gel is taken, then a position of a markeris marked. Then the gel is washed twice with water, and immersed in a0.25 M HCl solution for 15 minutes. Subsequently, the gel is immersed ina 0.4 N NaOH solution for 10 minutes while gently shaking. According toa conventional method, the gel is transferred to a filter, and after 4hours, the filter is collected and washed with 2×SSC twice. After thefilter is fully dried, baking (75° C., 3 hours) is carried out. Afterbaking, the filter is immersed in a 0.1×SSC/0.1% SDS solution andtreated at 65° C. for 30 minutes. Thereafter, the filter is immersed ina 3×SSC/0.1% SDS solution. The thus treated filter is placed in aplastic bag with a prehybridization solution. The mixture is treated at65° C. for 3 to 4 hours.

Thereafter, ³²P labeled probe DNA and a hybridization solution areplaced in the plastic bag, and the mixture is reacted at 65° C. forabout 12 hours. After completion of the hybridization, the filter iswashed under conditions of a suitable salt concentration, reactiontemperature and reaction time (e.g. 2×SSC, 0.1% SDS solution, roomtemperature, 10 minutes). The filter is placed in a plastic bag and asmall amount of 2×SSC is added thereto. The plastic bag is hermeticallysealed and autoradiography is carried out. The rearranged VDJ and VJgenes, which respectively encode the H chain and L chain of the mousemonoclonal antibody, are identified by the above described Southernblotting. A region comprising the DNA fragment identified by this methodis fractionated by cesium chloride density-gradient centrifugation, andthe obtained fraction is integrated into a phage vector (e.g. charon4A,charon28, λEMBL3, λEMBL4, etc.) Escherichia coli (e.g. LE392, NM539,etc.) is transformed with said phage vector so as to prepare a genomelibrary. Using a suitable probe (e.g. an H chain J gene, an L chain (κ)J gene, etc.), according to, for example, Benton-Davis method (Science,Vol. 196. pages 180 to 182, (1977)), plaque hybridization is carried outon the genome library so as to obtain a positive clone comprising eachof the arranged VDJ and VJ genes. The restriction enzyme map of theobtained clone is prepared, the nucleotide sequence is determined toconfirm whether a gene of interest comprising a rearranged V_(H) (VDJ)gene or V_(L) (VJ) gene can be obtained.

On the other hand, a human C_(H) gene and a human C_(L) gene used inchimerization are isolated separately. For example, in the case ofproducing a chimeric antibody with human IgG1, a Cγ1 gene as a C_(H)gene and a Cκ gene as a C_(L) gene are isolated. Since a mouseimmunoglobulin gene has high homology with a human immunoglobulin gene,the above two genes can be obtained by isolating from a human genomelibrary, using, as probes, a mouse Cγ1 gene and a mouse Cκ1 gene whichrespectively correspond to a human Cγ1 gene and a human Cκ gene.

Specifically, for example, using a 3 kb of HindIII-BamHI fragment from aclone Ig146 (Proc. Natl. Acad. Sci. USA, Vol. 75, pages 4,709 to 4,713(1978)) and a 6.8 kb of EcoRI fragment from a clone MEP10 (Proc. Natl.Acad, Sci. USA, Vol. 78, pages 474 to 478 (1981)) as probes, a DNAfragment comprising the human κ gene with an enhancer region is isolatedfrom the HaeIII-AluI genomic library of a human λCharon4A (Cell, Vol.15, pages 1,157 to 1,174 (1978)). In the case of the human Cγ1 gene, forexample, human fetal liver cell DNA is cleaved with HindIII, andfractionated by agarose gel electrophoresis. Thereafter, 5.9 kb of theobtained band is inserted into λ788 followed by isolation with the aboveprobes.

Using the thus obtained mouse V_(H) gene and mouse V_(L) gene, as wellas human C_(H) gene and human C_(L) gene, taking a promoter region andan enhancer region into consideration, the human C_(H) gene is placeddownstream of the mouse V_(H) gene and the human C_(L) gene is placeddownstream of mouse V_(L) gene; and these genes are then integrated intoan expression vector(s) such as pSV2gpt or pSV2neo, using suitablerestriction enzymes and DNA ligase according to conventional methods.Herein, a chimeric gene of mouse V_(H) gene/human C_(H) gene and achimeric gene of mouse V_(L) gene/human C_(L) gene may be simultaneouslyintegrated into a single expression vector, or may be integrated intotwo separate expression vectors.

The thus produced expression vector, into which the chimeric gene isinserted, is introduced into a myeloma cell such as P3X63.Ag8.653 cellor SP2/0 cell, which does not produce an antibody by itself, byspheroplast fusion. DEAE-dextran method, calcium phosphate method,electroporation or the others. The obtained cells are cultured in amedium which contains an agent for an agent-resistant gene introduced inthe expression vector so as to select a transformed cell, therebyobtaining a chimeric monoclonal antibody producing cells of interest.Then, a chimeric monoclonal antibody of interest is obtained from theculture supernatant of the thus selected antibody producing cell.

The “humanized antibody (CDR-grafted antibody)” of the present inventionis a monoclonal antibody produced by genetic engineering, and itspecifically means a humanized antibody characterized in that a part orthe entire CDRs of the hypervariable region is derived from a mousemonoclonal antibody, the framework region in the variable region isderived from human immunoglobulin, and the constant region is a humanimmunoglobulin region.

Herein, the CDRs of the hypervariable region means three regions (CDR1,CDR2 and CDR3) which exist in the hypervariable region of the variableregion of an antibody and complementarily bind to an antigen, and theframework region (FR) in the variable region means four regions (FR1,FR2, FR3 and FR4) which are located before and after the above threeCDRs and are relatively well conserved. In other words, it is a mousemonoclonal antibody, in which all the regions other than a part or theentire CDR of the hypervariable region are substituted by thecorresponding human immunoglobulin regions. The constant region derivedfrom the corresponding human immunoglobulin region has a specific aminoacid sequence depending on isotypes such as IgG, IgM, IgA, IgD and IgE.However, the constant region of humanized antibody of the presentinvention may be the constant region of human immunoglobulin whichbelongs to any type of the isotypes. Preferably, it is the constantregion of human IgG. Moreover, there is not a limit as to the FR in thevariable region derived from human immunoglobulin.

The humanized antibody of the present invention can be produced, forexample, as follows. However, needless to say, the production method isnot limited to the followings: For example, a recombinant humanizedantibody derived from a mouse monoclonal antibody can be produced bygenetic engineering, referring to Domestic Announcement No. Hei4-506458,Japanese Patent Laid-Open No. Sho62-296890, and others. The productionof a humanized antibody involves, first, selecting human V_(H) and V_(L)having high homology with the amino acid sequence in the V region of amonoclonal antibody of interest: searching for and selecting the aminoacid residues in FR region having an effect on the higher-orderstructure of CDR by computer modeling, and designing all amino acidsequences in V_(H) and V_(L) regions which consist of mouse-derived CDR(including a small amount of FR sequence) and a human-derived PR regionsequence. As a C region, the desired class or sub-class of a humanantibody is selected. The V_(H) and V_(L) genes are produced by chemicalsynthesis, PCR or site-directed mutagenesis. Taking a promoter regionand an enhancer region into consideration, the V region genes of the Hchain and L chain of the thus designed mouse antibody are ligated to theC region genes of the H chain and L chain of a human antibody,respectively. The obtained genes are integrated into expression vectorsseparately, and the vectors are then introduced into cells forexpression. As an expression vector, such as pSV2gpt or pSB2neo, isoften used. Expression may also be carried out using a single vector.Subsequently, the expression vector is then introduced into a cell line,such as mouse myeloma Sp2/0, which does not produce expression andsecretion of immunoglobulin. The Antibody may be generated not only in amyeloma but also in animal cells. insect cells, yeast or Escherichiacoli.

The “human antibody” of the present invention is immunoglobulin, inwhich all the constructed regions including the variable and constantregions of the H chain as well as the variable and constant regions ofthe L chain are derived from a gene encoding human immunoglobulin. Bythe same method as described above for the production of a polyclonal ormonoclonal antibody, the human antibody can be produced by the antigenicor immunogenic stimulation of a transgenic animal, which is produced byintegrating, for example, at least a human immunoglobulin gene into thegene locus of mammals other than human such as mouse according toconventional methods. For example, a transgenic mouse which produces ahuman antibody can be produced by methods described in Nature Genetics,Vol. 7, pages 13 to 21, 1994; Domestic Announcement No. 4-504365;International Publication WO94/25585; Nikkei Science, June, pages 40 to50, 1995: Nature, Vol. 368, pages 856 to 859, 1994: and DomesticAnnouncement No. Hei6-500233.

The term “portion of an antibody” or “fragment of an antibody” is usedherein to mean an antibody fragment comprising at least one variableregion, and means a partial region of the above described antibody,preferably the monoclonal antibody of the present invention.Specifically, it refers to Fv, F(ab′)₂, Fab′ or Fab. Each of the terms“F(ab′)₂” and “Fab′” is used herein to mean an antibody fragmentobtained by treating immunoglobulin (monoclonal antibody) with proteasesuch as pepsin or papain and digesting before or after a disulfide bondexisting between two H chains in a hinge region. For example, when IgGis treated with papain, it is cleaved upstream of the disulfide bondsbetween the two H chains in the hinge region, thereby producing twohomologous antibody fragments in which an L chain fragment consisting ofVL (an L chain variable region) and CL (an L chain constant region) andan H chain fragment consisting of VH (an H chain variable region) andCHγ1 (a γ1 region in an H chain constant region) are bound in aC-terminal region by a disulfide bond. Each of the two identicalantibody fragments is called Fab′. When IgG is treated with pepsin, itis cleaved downstream of the disulfide bonds between the two H chains inthe hinge region, thereby producing an antibody fragment which isslightly larger than the above described two Fab's which are bound eachother in the hinge region. This antibody fragment is called F(ab′)₂.

<Recombinant Vector and Transformant>

The present invention further provides a recombinant vector comprisingthe gene of the present invention or the DNA fragment thereof.

The recombinant vector can be simply produced by ligating a desired geneto a vector for cloning (e.g. plasmid DNA, etc.) available in thepresent field of the art according to conventional methods. Examples ofthe vector used include a plasmid derived from Escherichia coli such aspBluescript, pUC18, pUC19 or pBR322, but are not limited thereto.

For the purpose of producing a desired protein, an expression vector isparticularly useful. The type of the expression vector is notparticularly limited, as long as it has functions to express a desiredgene in various types of host cells such as procaryotic cells and/oreucaryotic cells so as to produce a desired protein. Examples of apreferred expression vector include expression vectors for Escherichiacoli such as pQE-30, pQE-60, pMAL-C2, pMAL-p2 and pSE420, expressionvectors for yeast such as pYES2 (Saccharomyces), pPIC3.5K, pPIC9K andpAO815 (supra; genus pichia), and expression vectors for insects such aspBacPAK8/9, pBK283, pVL1392 and pBlueBac4.5.

Examples of a method of inserting a DNA fragment of the gene of thepresent invention into a vector such as a plasmid include a methoddescribed in Sambrook, J. et al., Molecular Cloning, A LaboratoryManual, (second edition), Cold Spring Harbor Laboratory, 1.53 (1989) andthe like. Simply, a commercially available ligation kit (e.g. TakaraShuzo Co., Ltd.) can be used. The thus obtained recombinant vector (e.g.a recombinant plasmid) can be introduced into a host cell according to amethod as described below.

The recombinant vector of the present invention can be introduced(transformed or transfected) into a host cell according to previouslyknown methods. Examples of such a method include calcium chloride methoddescribed in Sambrook, J. et al., Molecular Cloning, A LaboratoryManual. (second edition), Cold Spring Harbor Laboratory, 1.74 (1989),calcium chloride/rubidium chloride method electroporation,electroinjection, a chemical treatment method such as PEG, a method ofusing gene gun and others. Otherwise, in a case where the host cell isbacterium (E. coli, Bacillus subtilis, etc.), methods such as Cohen etal.'s method (Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)), protoplastmethod (Mol. Gen. Genet., 168, 111 (1979)) or competent method (J. Mol.Biol., 56, 209 (1971)) can be applied to introduce a recombinant vectorinto the host cell. In a case where the host cell is Saccharomycescerevisiae, methods such as Hinnen et al.'s method (Proc. Natl. Acad.Sci. USA, 75, 1927 (1978)) or lithium method (J. Bacteriol., 153, 163(1983)) can be applied, and in a case where the host cell is a plantcell, methods such as leaf disk method (Science, 227, 129 (1985)) orelectroporation (Nature, 319, 791 (1986)) can be applied. In the case ofan animal cell, methods such as Graham's method (Virology, 52, 456(1973)) can be applied, and in the case of an insect cell, methods suchas Summers et al.'s method (Mol. Cell. Biol., 3, 2156-2165 (1983)) canbe applied.

A host cell used to produce a tranformant is not particularly limited,as long as it is suitable for the recombinant vector of the presentinvention and can be transformed, and various cells such as native cellscommonly used in the technical field of the present invention orartificially established recombinant cells can be used. Examples of thehost cell used include procaryotic cells such as bacteria (Escherichia,Bacillus, etc.); lower eucaryotic cells including a unicellular hostsuch as yeast (Saccharomyces, genus pichia, etc.): higher eucaryoticcells such as silk worm; and others. Examples of a preferred host cellinclude Escherichia coli, yeast, an insect cell and others. Specificexamples of such a host cell include Escherichia coli (M15, JM109, BL21,etc.), yeast (INVScl (Saccharomyces), GS115 and KM71 (supra; genuspichia), etc.), insect cells (BmN4, silk worm larva, etc.), and others.Examples of an animal cell include a mouse-derived, Xenopus-derived,rat-derived, hamster-derived, monkey-derived or human-derived cell, aculture cell line established from these cells, and others.

When bacteria, especially Escherichia coli, is used as a host cell, thecorresponding expression vector is generally comprised of at least apromoter/operator region, an initiation codon, a gene encoding a desiredprotein, a termination codon, a terminator and a replicable unit. Whenyeast, a plant cell, animal cell or insect cell is used as a host cell,generally, the corresponding expression vector preferably comprises atleast a promoter, an initiation codon, a gene encoding a desiredprotein, a termination codon and a terminator. Moreover, a host cell mayalso comprise DNA encoding a signal peptide, an enhancer sequence, thenon-translation regions of the 5′-side and 3′-side of a desired gene, aselective marker region, a replicable unit and others, as appropriate.

An example of a preferred initiation codon for the vector of the presentinvention includes a methionine codon (ATG). An example of a terminationcodon includes a commonly used termination codon (e.g. TAG, TGA, TAA.etc.) The replicable unit means DNA capable of replicating all the DNAsequences thereof in a host cell, and examples include a native plasmid,an artificially modified plasmid (which is pr pared from a nativeplasmid), a synthetic plasmid and others. Examples of a preferredplasmid include a plasmid pQE30, pET, pCAL or an artificially modifiedproduct of these plasmid (i.e. a DNA fragment obtained by treatingpQE30, pET or pCAL with suitable restriction enzymes) for Escherichiacoli; a plasmid pYES2 or pPIC9K for yeast; and a plasmid pBacPAK8/9 orthe others for an insect cell.

As an enhancer sequence and a terminator sequence, products such as onederived from SV40, which are commonly used by a person skilled in theart, can be used. As a selective marker, commonly used products can beused according-to conventional methods. Examples of such a selectivemarker include resistance genes to antibiotics such as tetracycline,ampicillin, kanamycin, neomycin, hygromycin, spectinomycin orchloramphenicol.

An expression vector can be prepared by continuously and circularlyligating at least the above promoter, initiation codon, gene encoding adesired protein termination codon and terminator region to a suitablereplicable unit. This time, a suitable DNA fragment (e.g. a linker,another restriction site, etc.) can also be added, as desired, byapplying conventional methods such as digestion with restriction enzymesor ligation-with T4DNA ligase.

<Receptor, Screening Method>

It is considered that the protein encoded by the gene of the presentinvention acts as an entrance to induce or stimulate the secretion ofG-CSF (that is, as a possibility, a model can be speculated, in which anexternal ligand is bound to the protein of the present inventionexisting on the surface of a macrophage, a signal generated by the eventis transmitted into the cell so that the macrophage releases G-CSF, butthe present invention is not restricted to the above theory.)Accordingly, the protein of the present invention can be a receptor of aG-CSF secretion inducing or stimulating factor or a portion thereof. Theterm “a portion of a receptor” is used herein to include a receptorportion which is a part constituting the receptor modified with a sugarchain or the like. It is considered that this receptor has a bindingability (which is referred to also as affinities) for a substancecapable of inducing the generation of G-CSF, such as a monoclonalantibody or a fragment thereof produced from hybridoma deposited underaccession No. FERM BP-6103; and that the receptor exists in the cellmembrane of a cell capable of generating G-CSF such as a macrophage. Thepresent invention provides the above receptor.

The present invention further provides a method of screening a usefulsubstance, which is characterized in that the protein or receptor of thepresent invention is used. The screening method of the present inventioncomprises the steps of (a) bringing a substance into contact with theprotein according to any one of claims 9 to 12 or a cell comprising thereceptor according to claim 20; and (b) determining the effect of thesubstance obtained through the contact with the protein or receptor. Thepresent screening method further comprises the steps of determining theaffinity of a substance in question (simply referred to as “a substance”or “a test compound” at times) for the protein of the present invention,the above described receptor, or a cell comprising the same (e.g.analysis of the affinity between a cell having the receptor of thepresent invention on a surface thereof and the substance in questionusing a flow cytometer); determining the effect of the substance inquestion obtained through the contact with the above receptor (e.g. thegeneration of G-CSF from a macrophage, the generation of a markersubstance from a suitable transformed cell); or comparing the structureof the substance in question (e.g. when the substance in question is aprotein, it is the amino acid sequence) with the structure of theprotein of the present invention (e.g. the amino acid sequence).

Moreover, the screening method of the present invention includes meansof designing a compound with a computer on the basis of the structuralinformation of the protein or receptor of the present invention,synthesizing many types of the thus designed compounds and/or analogsthereof by a technique such as combinatorial chemistry, and selecting auseful substance from the synthesized compounds and/or analogs thereofusing a suitable technique (e.g. HTS (high throughput screening, etc.);means of synthesizing and selecting more modified products, using such asubstance as a lead compound; and others.

The protein or receptor of the present invention used in the screeningis, preferably, (a) a protein having an amino acid sequence shown in SEQID NO: 4; (b) a protein having an amino acid sequence comprising adeletion, substitution, addition or insertion of one or several aminoacids with respect to the amino acid sequence shown in SEQ ID NO: 4, andhaving a binding ability for an antibody capable of inducing andsecreting a G-CSF or a fragment thereof: (c) a protein having 50% ormore (preferably 60% or more, more preferably 70% or more, furtherpreferably 80% or more, further more preferably 90% or more,particularly preferably 94% or more, and most preferably 98% or more)homology with the amino acid sequence shown in SEQ ID NO: 4, and havinga binding ability for an antibody capable of inducing and secreting aG-CSF or a fragment thereof: (d) a protein being encoded by DNA whichhybridizes with DNA having the nucleotide sequence shown in SEQ ID NO: 3under stringent conditions, and having a binding ability for an antibodycapable of inducing and secreting a G-CSF or a fragment thereof: or areceptor having any one of the above proteins.

A more specific example of the screening method is as follows: A vectorinto which a G-CSF promoter gene; a gene encoding a marker protein suchas luciferase, β-galactosidase, a green fluorescence protein (GFP),β-lactamase or chloramphenicol acetyl transferase (CAT), which locatesdownstream of the above gene; and an agent resistance gene to agentssuch as tetracycline, ampicillin, kanamycin, neomycin, hygromycin orspectinomycin, which locates further downstream of the above gene, areinserted, is constructed. This vector is introduced into a cell havingthe receptor including the protein of the present invention (e.g. amacrophage cell line, preferably a human-derived macrophage cell line).The obtained cell is treated in a medium containing an agent to select acell group which forms a colony. Thereafter, a clone expressing a markerprotein is further selected using a G-CSF inducing agent such aslipopolysaccharide (LPS). Further, it is confirmed that the expressionof the marker protein reflects the expression of G-CSF mRNA. The thusobtained transformed cell line is treated with various substances sothat a substance inducing the expression of the marker protein isscreened.

In the screening method of the present invention, a portion of theprotein of the present invention, which is recognized by an antibodyhaving induction or secretion promotion activity of G-CSF (e.g. amonoclonal antibody produced from hybridoma deposited under accessionNo. FERM BP-6103) or a fragment thereof, is particularly important insome cases. A method of determining such an important portion is wellknown to a person skilled in the art. For example, the present inventorshave determined a portion recognized by a monoclonal antibody producedfrom hybridoma deposited under accession No. FERM BP-6103 in one proteinof the present invention which has an amino acid sequence shown in SEQID NO: 2 (refer to Example 9).

<Novel Substance>

The useful substance obtained by screening is a substance capable ofchanging the production of G-CSF. This substance includes; (a) asubstance having a binding ability to a receptor (it is predicted that,as a result of binding, the substance changes the receptor and transmitsinformation into a cell through the receptor), and inducing theproduction of G-CSF (referred to also as an agonist or stimulant); (b) asubstance having a binding ability to a receptor (It is predicted that,as a result of binding, the substance antagonizes the binding of asubstance inducing the production of G-CSF to a receptor and inhibitsthe stimulation), and not inducing the production of G-CSF (referred toalso as an antagonist or blocking agent); and (c) a substance having abinding ability to a receptor (it is predicted that as a result of thebinding, the substance inhibits the binding of a substance inducing theproduction of G-CSF to a receptor), and inhibiting the activity ofproducing G-CSF-of a receptor itself (referred to also as an inverseagonist or counteragent).

This substance is novel. Therefore, the present invention provides: (a)a substance having a binding ability to a receptor (it is predictedthat, as a result of binding, the substance changes the receptor andtransmits information into a cell through the receptor), and inducingthe production of G-CSF; (b) a substance having a binding ability for areceptor (it is predicted that, as a result of binding, the substanceantagonizes the binding of a substance inducing the production of G-CSFto a receptor and inhibits the stimulation), and not inducing theproduction of G-CSF: or (c) a substance having a binding ability for areceptor (it is predicted that, as a result of the binding, thesubstance inhibits the binding of a substance inducing the production ofG-CSF to a receptor) and inhibiting the activity of producing G-CSF of areceptor itself, wherein the above substance is obtained by thescreening method of the present invention. Moreover, the presentinvention further provides a substance having a binding ability for theprotein or receptor of the present invention, which is selected from agroup consisting of: (a) a substance which induces the generation ofG-CSF (it is predicted that, as a result of binding the substancechanges the receptor and transmits information into a cell through thereceptor); (b) a substance which does not induce the production of G-CSF(it is predicted that, as a result of binding, the substance antagonizesthe binding of a substance inducing the production of G-CSF to areceptor and inhibits the stimulation): and (c) a substance whichinhibits the activity of producing G-CSF of a receptor itself (it ispredicted that, as a result of the binding, the substance inhibits thebinding of a substance inducing the production of G-CSF to a receptor).Hereinafter, these substances may be called “the substance of thepresent invention” at times. The technical scope of the substance of thepresent invention does not include known substances.

Examples of the substance of the present invention include the antibodyof the present invention; a fragment thereof; or other low molecularcompounds, which induce the production of G-CSF, which antagonize thebinding of a substance inducing the production of G-CSF to a receptorand inhibit the stimulation, or which inhibit the action to produceG-CSF of a receptor itself and also inhibit the production of G-CSF.

With regard to the above “other low molecular compounds,” many types oflow molecular compounds can be synthesized by means well known to aperson skilled in the art such as combinatorial chemical synthesis, oran established chemically synthetic library can also be used (M. J.Plunket et al., Development of New Drugs and Combinatorial Chemistry:Nikkei Science 7, 62-69 (1997); Combinatorial Chemistry, edited by JapanCombinatorial Chemistry Focus Group, Kagaku Dojin Publishing Co., Inc.(1998)).

When a substance in question is an antibody, the affinity for (orinhibition of the binding to) the above receptor can be determined byanalyzing a macrophage cell line binding to the antibody by flowcytometry or ELISA, or by equivalent methods.

An action to induce (or inhibit) the production of a G-CSF can bedetermined by a method described in Japanese Patent Laid-Open No.Hei11-106400. The outline of the method will be described below.

A G-CSF promoter gene is inserted into the restriction sites betweenXhoI and NcoI of a PicaGene Enhancer Vector 2 (Wako Pure ChemicalIndustries, Ltd.), a luciferage gene is ligated downstream of it-insteadof the G-CSF gene, and further a neomycin-resistance gene cut frompMC1Neo Poly A is inserted into the SalI site which is locateddownstream of SV40, so that a PicaGCSFneo vector is produced. Thisvector is introduced in the RAW264.7 cell by electroporation. Theobtained gene is treated with a medium containing geneticin, and cellsforming a colony are selected. Thereafter, a clone showing luciferaseactivity is selected among the geneticin-resistant clones using a G-CSFinducing agent such as LSP. It is confirmed that the luciferase activityreflects the expression of G-CSF mRNA, by Northern blotting analysis,using ³²P labeled mouse G-CSF cDNA as a probe. The thus obtainedtransformed macrophage cell line is inoculated in an amount of 5×10⁴cells per well of a 96-well microplate followed by culture at 37° C. for24 hours. Thereafter, the cells are treated with the previously obtainedagonist or antagonist as necessary, and a substance in question is thenadded thereto in a concentration of 0, 3.75, 7.5, 15, 30 and/or 60μg/ml. After culture at 37° C. for 18 hours, luciferase activity isdetermined.

Otherwise, the action to induce (or inhibit) the production of G-CSF canalso be determined by bioassay for G-CSF, which is described in Example14 hereafter.

Whether or not it is the substance of the present invention can bedetermined by various criteria. For example, (1) a system capable ofproducing G-CSF, or a system showing a parameter capable of reflectingthe production of G-CSF is constructed, then, a group with the additionof a test compound and a suitable control group (e.g. a system using aknown substance instead of the test compound, or a system using neitherthe test compound nor the alternative) are prepared, and determinationcan be made using the value obtained in the control group as a standard.Herein, a group comprising both the test compound and an agonist orantagonist, which has previously been obtained, may also be prepared.Moreover, (2) determination can also be made on the basis of the bindingability between the receptor or the protein of the present invention andthe test compound (e.g. the affinity constant (referred to also as Km,association constant) between the receptor or protein and the testcompound, which is determined by appropriate means). In this case, thevalue obtained using a known substance instead of the test compound canbe used as a standard. Otherwise, (3) determination can also be made bycombination of (1) and (2).

More specifically, when a cell having the protein of the presentinvention on a surface thereof and is transformed so that the luciferaseactivity can reflect the actual expression of G-CSF mRNA is stimulatedwith a test compound, and the maximum luciferase activity obtained underappropriate conditions is higher than the control group, preferably itis about 1.01-fold or more, more preferably it is about 2-fold or more,further preferably about 20-fold or more, and most preferably about60-fold or more, the test compound can be defined as “a substance havinga binding ability for the protein or receptor of the present invention,and (a) inducing the production of G-CSF” in the present invention (seeExamples). Moreover, a system which comprises a group allowing thepreviously obtained agonist or antagonist to co-exist with the testcompound can be used to determine whether or not it is “a substancehaving a binding ability for the protein or receptor of the presentinvention, and (b) not inducing the production of G-CSF: or (c) inducingthe production of G-CSF” in the present invention.

The substance of the present invention may also induce cytokines otherthan G-CSF such as interleukin (IL), interferon (INF), tumor necrosisfactor (TNF) or various colony-stimulating factors (CSFs). A substanceinducing G-CSF more selectively than other cytokines is one preferredembodiment of the substance of the present invention. An example of sucha substance includes a substance, which induces other cytokines at alevel of less than about 10-fold of the control, but induces G-CSFcytokine at a level of about 10-fold or more, preferably about 20-foldor more, and more preferably about 40-fold or more of the control, whenit is used in an appropriate concentration. The induction of othercytokines can be determined by a method well known to a person skilledin the art.

<Use of the Gene and others of the Present Invention as aPharmaceutical>

The gene of the present invention can be used for diagnosis, preventionand treatment (e.g. gene therapy, etc.) of diseases with which one typeof the leukocytes, neutrophils, is associated (e.g. neutropenia, etc.),or diseases relating to blood cells such as erythrocyte, leukocyte orthrombocyte. Moreover, the inventive protein or a partial peptidethereof, antibody or a fragment thereof, ligand, receptor, or substance(hereinafter, these may be generically referred to as “the protein andothers of the present invention”) can be used as a pharmaceutical whichcontrols the number of neutrophils in the blood or bone marrows, or morewidely, controls the number of blood cells such as erythrocytes,leukocytes or thrombocytes. That is to say, the gene, protein and othersof the present invention can be used for the treatment of neutropenia asa side effect of an anticancer agent or neutropenia occurring afteroperation of bone marrow transplantation, or cytopenia regarding thereduction of the number of erythrocytes, leukocytes, thrombocytes andothers, and the diagnosis, prevention and treatment of aplastic anemia.

Moreover, the present inventors have found that the protein or receptorof the present invention is associated with the induction of theproduction of G-CSF. Accordingly, a substance having a binding abilityto the protein or receptor of the present invention can be used as apharmaceutical promoting the production of G-CSF or controllingbiological activity regarding G-CSF. This substance can be usedespecially for the treatment of neutropenia as a side effect of ananticancer agent, neutropenia occurring after operation of bone marrowtransplantation, the cytopenia regarding the reduction of the number oferythrocytes, leukocytes, thrombocytes and others, or neutropeniaoccurring after bone marrow transplantation, and the diagnosis,prevention and the treatment of aplastic anemia.

Usually, the protein and others of the present invention can beadministered systemically or locally, generally in a parenteral form. Ofparenteral administrations, an intravenous administration isparticularly preferable.

The gene of the present invention can be administered systemically orlocally by what is called gene therapy in which a gene is introducedinto a cell in vivo or in vitro. The gene transfer can be carried outby, for example, the method described in Blomanual UP Series, BasicTechniques of Gene Therapy, edited by Takashi Shimada, Izumi Saito andTakaya Ozawa, published by Yodosha Co., Ltd., 1996. When a gene isintroduced into a cell in vitro, methods of using a retrovirus vector,an adenovirus vector, an adeno-associated virus (AAV) vector, cationicliposome or HVJ liposome; calcium phosphate method; DEAE dextran method;and others can be used. When a gene is introduced into a cell An vivo,methods of using a retrovirus vector, an adenovirus vector, anadeno-associated virus (AAV) vector, cationic liposome or HVJ liposomecan be used.

Dosage is different depending on age, sex, body weight, symptomconditions, treatment effect, administration route, treatment time or anagent to be administered (the type of a protein or gene), but the agentcan be administered in a range of 1 μg to 100 g, preferably 10 μg to1000 mg per adult per time, one to several times per day by parenteraladministration. Since the dosage is changed by various conditions, insome cases, an amount smaller than the above range of dosage may besufficient, but in other cases, a dosage over the above range may berequired. Examples of injections for parenteral administration of thepresent invention include aseptic aqueous or non-aqueous solution,suspension, emulsion and others. The aqueous or non-aqueous solution orsuspension is obtained by mixing one or more active substances with atleast one inactive diluent. Examples of the aqueous diluent includedistilled water for injection, physiological salt solution and others.Examples of the non-aqueous diluent include propylene glycol,polyethylene glycol, vegetable oil such as olive oil, alcohols such asethanol, and others.

This composition may also comprise an adjuvant, such as an antiseptic,wetting agent, emulsifier, dispersing agent or stabilizer (e.g.arginine, aspartic acid, etc.)

The injections are sterilized by filtration through a bacteria retainingfilter, with mixing of germicide, or irradiation. Moreover, it is alsopossible to produce an aseptic solid composition, for example, byfreeze-drying, and dissolve it in aseptic distilled water for injectionor other solvents b fore use.

Examples of other compositions for parenteral administration include aliquid for external use prescribed by conventional methods, suppositoryand pessary for enteric administration, and others, which comprise oneor more active substances.

The present invention is further described in the following examples.The examples are provided for illustrative purposes only, and are notintended to limit the scope of the invention.

EXAMPLES Example 1 Production and Purification of 3-4H7 Antibody(Produced from Hybridoma Deposited Under Accession No. FERM BP-6103;Described in Japanese Patent Application No. 9-266591 Specification

All the steps of the production and purification of 3-4H7 (IgM), asignaling antibody (agonist antibody), were carried out by asepticmanipulation to prevent contamination of microorganisms and endotoxin,such as lipopolysaccharide (LPS). Moreover, the treatment was carriedout, while always measuring the endotoxin concentration of the culturesolution and reagent used (described later) and checking that theconcentration was within the tolerance (0.1 or less EU/ml). That is, inan Integra CL1000 culture flask, hybridoma cells were suspended in anASF104 serum free medium at a density of 1×10⁸ cells per ml, and culturewas carried out for 5 days. The obtained supernatant was diluted with a3-fold amount of 10 mM phosphate buffer (pH 6.8) containing 200 mM NaCl.It was loaded on an MGPP column which was previously equilibrated, andwashed with a 10 mM phosphate buffer (Ph 6.8) containing 200 mM NaCl toeliminate contaminants. An antibody was eluted with a 300 mM phosphatebuffer (pH 6.8), and the obtained antibody was dialyzed in a phosphatebuffered saline (PBS, pH 7.4) to obtain an antibody solution. The purityof the purified antibody was determined by FPLC and SDS-PAGE, and thenthe antibody was subjected to the following experiment.

Example 2 Quantitation Assay for LPS Concentration

The concentration of LPS contaminated in the solutions used in theexperiment was assayed by Limulus method using an Endospecy ToxicolorSystem (Seikagaku Kougyo Corporation). That is to say, 50 μl of thesolutions were placed on an endotoxin free 96-well microplate, 50 μl ofa lysate-synthetic reaction substrate solution was added thereto on ice,immediately followed by reaction at 37° C. for 30 minutes. Immediatelyafter that, 50 μl each of a sodium nitrite solution, ammonium sulfamatesolution, N-(1-naphtyl)ethylenediaminedihydrochloride-N-methyl-2-pyrrolidone solution were added thereto inthis order, and then absorption at 550 nm was measured using MicroplateReader M-Tmax (Molecular Device Corp.). By subtracting the absorptionvalue at 650 nm, a control value, LPS concentration was calculated bySoftMax 1.5 program. A USP standard product was used as standardconcentrations of LPS, and the same LPS was used when cells werestimulated.

Example 3 Cloning of Antigen Gene Against 3-4H7 Antibody in theMacrophage Cell Lines

(1) Preparation of Poly A⁺ RNA From the Macrophage Culture Cell Line,RAW264.7

Approximately 0.3 mg of a total RNA was prepared from the RAW264.7 cells(2×10⁸ cells) by guanidium thiocyanate/phenol chloroform single stepextraction (Labobanual Gene Engineering, 3^(rd) edition, 83-84, 1996).The obtained total RNA was further purified using an oligo(dT) cellulosecolumn (Life Technologies) to obtain approximately 5 μg of poly A⁺ RNA.

(2) Construction of cDNA Library

The synthesis of cDNA was carried out by linker-primer method(modification of Gubler-Hoffmann method: Gene, 25:263-269, 1983), usinga ZAP-cDNA synthesis kit from STRATAGENE. That is, a linker-primer (2.8μg) containing olido(dT)₁₈ and a XhoI recognition sequence and reversetranscriptase (MMLV-RTase; 70 units) were added to the poly A⁺ RNA (5μg) obtained in (1) as described above which was followed by reaction at37° C. for 60 minutes to synthesize complementary single stranded DNA(ss-cDNA). At the time, 5-methyl dCTP was taken therein so as to protectcDNA from the subsequent restriction enzyme treatment. Thereafter, RNaseH (2 units) was acted thereon, so that a nick was generated on a DNA-RNAhybrid. Using the generated RNA fragment as a primer, E. Coli DNApolymerase I (100 units) was added thereto followed by reaction at 16°C. for 150 minutes to synthesize ds (double stranded)-cDNA (8 μg). Aftercarrying out phenol/chloroform extraction and ethanol precipitation, thethus prepared ds-cDNA was reacted at 72° C. for 30 minutes in a reactionsolution comprising pfu DNA polymerase (5 units) for blunt-ending.

Phenol/chloroform extraction and ethanol precipitation were carried outagain, an ScoRI adaptor (0.35 μg) which was previously annealed in abuffer containing T4 DNA ligase (4 units) was added thereto by reactingat 8° C. overnight, and then the EcoRI side of the cDNA wasphosphorylated by reacting with T4 polynucleotidekinase (10 units) at37° C. for 30 minutes. Thereafter, the linker-primer portion was cleavedby reaction with XnoI (120 units) at 37° C. for 90 minutes, the obtainedcDNA was fractionated by size using a spin column, and it was confirmedby 1% agarose gel electrophoresis that the length of the DNA strand was0.5 kbp or longer. Thereafter, the DNA was reacted in a buffercontaining T4 DNA ligase (4 units) at 12° C. overnight so that it wasinserted into a λZAP II vector (1 μg). The ligated λDNA was incubated atroom temperature for 90 minutes using a Gigapack III Gold packagingextract from STRATAGENE for in vitro packaging, it was then used toinfect Escherichia coli XL1-Blue MRF′, then a plaque was formed on aplate at 37° C. for 8 hours in the presence of IPTG(isopropyl-β-D-thiogalactopyranoside; 2.5 HM) and X-Gal (4 mg/ml), andthen titration was carried out. As a result, it was found that this cDNAlibrary comprised 1.2×10⁶ original clones. The cDNA library wasamplified with Escherichia coli XL1-Blue MRF′ to a size of 3.4×10⁹ pfu,and then used in the following screening.

(3) Screening of Genes Encoding Protein Having Affinity for 3-4H7Antibody

In the cDNA library constructed in (2) above, cDNA is introduced in the3′ side of the structural gene of β-galactosidase controlled by a lacpromoter. Accordingly, the cDNA is expressed as a fusion protein withβ-galactosidase. Thus, this fusion protein was blotted on the membrane,and immunoscreening was carried out using the 3-4H7 antibody as a probefor expression cloning by Escherichia coli. That is to say, 3.5×10⁴ pfueach of phages were mixed with top agarose, and the mixture wasinoculated on a plate with a diameter of 150 mm so as to prepare 20plates in total. These plates were incubated at 42° C. for 4 hours so asto form a plaque with a diameter of approximately 0.5 mm, anitrocellulose membrane in which sterile IPTG was previously immersedwas placed on the plaque, and incubation was carried out at 37° C. for 3hours to induce the expression. Thereafter, the membrane was removed,and blocking was carried out for 1 hour in a TBS-T solution (0.1% Tween20, 20 mM Tris buffer physiological salt solution, pH 7.6) containing 5%skim milk. Thereafter, the membrane was washed with TBS-T, and the 3-4H7antibody (1.6 μg/ml-1% BSA-TBS) as the first antibody was reactedtherewith for 1 hour. After washing, alkaline phosphatase labeledanti-mouse IgM rabbit antibody (Zymed, 0.6 μg/ml) as the second antibodywas reacted therewith for 1 hour. After the washing well again,incubation was carried out in a substrate solution (500 μg/ml NBT(nitroblue tetrazolium) containing 5 mM MgCl₂, 500 μg/mlBCIP(5-bromo-4-chloro-3-indolyl phosphate) TBS'solution, pH 9.5) in darkfor 30 minutes so as to develop color. The filter was placed togetherwith the master plate, so that a positive clone was collected from thetop agarose and amplified. Thereafter, the second, third and fourthscreenings were carried out in the same operations as described above.As a result, 22 positive clones were obtained from 7×10⁵ phages by thefirst screening, and finally 3 positive clones (MMR10, MMR17 and MMR19)were obtained.

(4) Analysis of Gene Sequences of the Obtained Positive Clones

In the λZAP II phage vector, a plasmid vector Bluescript SK (−) as awhole is inserted between the initiator region and the terminator regionof an f1 phage. Accordingly, a recombinant Bluescript is automaticallycut out by infecting with a helper phage, and a cloned DNA fragment canbe subcloned into the Bluescript. Thus, Escherichia coli XL1-Blue MRF′was infected with the three positive phages obtained in (3) above, andthen the obtained product was infected with a helper phage forsubcloning into pBluescript SK (−). This plasmid was transfected intoEscherichia coli SOLR to obtain approximately 20 μg of plasmid DNA. Thesequence of the plasmid DNA was analyzed by Primer Walking method. Usingan ABI PRISM BigDye Primer Cycle Sequencing Core Kit (PE), the purifiedplasmid DNA was subjected to sequence reaction with an M13Rev primer anda −21M13 primer, and then using a long range gel, the DNA s quence wasdetermined with an ABI 377 sequencer. As a result of the analysis, itwas found that the MMR19 clone had a 840 bp full-length cDNA nucleotidesequence which comprised the open reading frame of the protein. Incontrast, such an open reading frame was not confirmed in other clones.The nucleotide sequence of the MMR19 clone is shown in SEQ ID NO: 1.

(5) Primary Structure of Protein Predicted from Nucleotide Sequence ofthe cDNA Clone

The primary structure (as shown in SEQ ID NOS: 1 and 2) of the protein(hereinafter referred to as “MMRP19 protein”), which was predicted fromthe nucleotide sequence of the gene (MMR19) analyzed in (4) above, wascomposed of 241 amino acid residues, and the predicted molecular weightwas about 26.9 kDa.

Example 4 Comparison Between Mouse-derived MMR19 Gene and otherHomologous Genes by Database Search

With regard to the nucleotide sequence and the amino acid sequence shownin SEQ ID NO: 1, which were determined in Example 3, database search wascarried out by the programs, such as BLAST, EMBC and PROSITE, and thepresence or absence of homologous genes in human was analyzed both atamino acid residue level and at DNA level (GenBank, and DNA DATA BANK ofJAPAN (DDBJ), Ministry of Education, Culture, Sports, Science andTechnology. National Institute of Genetics, The Center for InformationBiology). The obtained results are shown in the following Tables 1 and2. As a result, it was shown that a gene highly homologous to the MMR19,the g ne of the present inv ntion, exists on human chromosome 9.

TABLE 1 Homology at amino acid level Position in amino acid sequenceHomology with human shown in SEQ ID NO: 1 homologue Amino acids 1 to 9183/91 (91%) Amino acids 50 to 146 83/97 (85%) Amino acids 1 to 78 70/78(89%) Amino acids 200 to 241 40/42 (95%) Amino acids 172 to 241 67/70(95%) Amino acids 103 to 150 46/48 (95%) Amino acids 169 to 241 58/73(79%)

TABLE 2 Homology at DNA level Position in nucleotide sequence Homologywith human shown in SEQ ID NO: 1 homologue Nucleotides 519 to 736189/218 (86%) Nuoleotides 666 to 689 23/24  (95%) Nucleotides 381 to 40322/23  (95%) Nucleotides 709 to 727 19/19  (100%)

Example 5 Coning of MMR19 Human Homologous Counterpart

Total RNA derived from a human normal brain tissue (Invitrogen) wassubjected to an oligo(dT) cellulose column to purify poly A⁺ RNA.Thereafter, according to the method shown in Example 3 (2), cDNA wassynthesized from the poly A⁺ RNA. With this cDNA as a template, PCRreaction was carried out using primers (a sense primer of positions 4 to22; 5′ CCATGTCTGGCTGTCAAGC-3′ (SEQ ID NO: 5); an antisense primer ofpositions 721 to 701; 5′-CCATTTTCTCCAACTGGGAGC-3′ (SEQ ID NO: 6)), whichwere prepared from a mouse antigen gene MMR19 sequence. As a result, apartial cDNA of an MMR19 human homologous counterpart was obtained.Subsequently, a full-length cDNA was obtained from the partial cDNAsequence of the obtained human homologous counterpart by a RACE method(using Marathon cDNA Amplification Kit from Clontech). That is, a cDNAlibrary derived from human normal brain tissue was blunt-ended, and aMarathon cDNA Adaptor (including an AP1 primer sequence) was ligatedhereto. Then, 5′-RACE PCR reaction was carried out using AP1 and GSP(Gene-Specific Primer) 1 (an antisense primer of positions 189 to 167:5′-AATTCCTCCTCCAGTCCCAGTGA-3′ (SEQ ID NO: 7)), and 3′-RACE PCR reactionwas carried out using GSP2 (a sense primer of positions 630 to 653:5′-TGGAGTATATGTGTGGGGGGAAAC-3′ (SEQ ID NO: 8)) and AP1, so that thesequences on 5′- and 3′-terminal sides were amplified in both reactions.When these PCR products were subjected to agarose gel electrophoresis, asingle band was observed regarding each PCR product. Accordingly, thiswas subjected to subcloning and then sequencing so as to decode thesequences of the 5′- and 3′-terminal sides. A sense primer(5′-AAGCCGTGCGGAGATTGGAGG-3′ (SEQ ID NO: 9); positions 1 to 21) wasproduced from the obtained 5′-RACE fragment, and an antisense primer(5′-GTCAGAAGAGATTCAGGGTGACC-3′ (SEQ ID NO: 10); positions 924 to 902)was produced from the obtained 3′-RACE fragment. With these primers, PCRreaction was carried out using the above used human normal braintissue-derived cDNA library as a template, and the PCR product wassubjected to T/A cloning using an AdvanTAge PCR Cloning Kit fromClontech so as to clarify the nucleotide sequence of the full-lengthcDNA of a human-type homolog comprising 1,136 bp open reading frame. Theobtained nucleotide sequence is shown in SEQ ID NO: 3. Using Genetyx-Mac(Software Development Co., Ltd.), the homology between the humanhomologous counterpart cDNA and the mouse MMR19 cDNA (840 bp) wasanalyzed. As a result, it was found that these cDNAs have 85.0% homologywith each other.

The primary structure of the protein predicted from the obtainednucleotide sequence of the MMR19 human homolog was comprised of 242amino acid residues, which is shown in SEQ ID NOS: 3 and 4. Thepredicted amino acid sequence had 93.8% homology with that of mouse.

Example 6 Analysts of the MMRP19 Protein

Rabbit polyclonal antibodies APA1, APA2 and APA3 against three types ofpeptides consisting of the partial amino acid sequences of the MMRP19protein were prepared as follows. That is to say, using a peptidesynthesizer (Applied Biosystems 433 type), peptides corresponding topositions 12 to 25, 58 to 71, and 228 to 241 of the amino acid sequenceof the protein predicted from the MMR19 were synthesized by9-Fluorenylmethoxycarbonyl (FMOC) method, and the peptides were thenpurified by reverse phase chromatography (Shimadzu LC8A type), so thatapproximately 25 mg each of three types of peptides was finallyobtained. The purified peptide was bound to a carrier protein hemocyanin(KLH) by an N-(6-maleimidocaproylxy)-succinimide crosslinking agent,each of the obtained product (1 mg) was mixed with Freund's completeadjuvant, and the obtained solution was immunized to a rabbit (female, 2to 2.5 kg) by a subcutaneous injection into the dorsal subcutis aboutthree times. After checking the antibody titer in the blood by ELISA,100 ml of the blood was collected from each rabbit to prepare serum.Further, each anti-peptide antibody (APA1, APA2 and APA3) was obtainedfrom the obtained serum using an IgG affinity column. APA1 is anantibody against a polypeptide having a sequence consisting of aminoacids 12 to 25 of the amino acid sequence (SEQ ID NO: 2) of the MMRP19protein. APA2 is an antibody against a polypeptide having a sequenceconsisting of amino acids 58 to 71 of the same sequence, and APA3 is anantibody against a polypeptide having a sequence consisting of aminoacids 228 to 241 of the same sequence.

Using these anti-peptide antibodies and the 3-4H7 antibody, the RAW264.7cell lysate was subjected to Western blot analysis. As a result, asshown in FIG. 1, all the bands recognized with any of these antibodieshad a molecular weight of 30.4 kDa, and therefore it was confirmed thatthese antibodies are bound to the same protein, that is, the MMRP19protein. It is considered that the difference between the thus obtainedmolecular weight (30.4 kDa) and the molecular weight calculated from theamino acid sequence (26.9 kDa) would be caused by sugar chainmodification.

Subsequently, the MMRP protein was subjected to hydropathy analysisaccording to Hoop and Woods method (Hoop, T. K. and Woods, K. P.: Mol.Immunol. 20, 483-489 (1983)). As a result, as shown in FIG. 2, theMMRP19 protein is likely to have two hydrophobic regions.

Thereafter, using the anti-peptide antibodies APA1, APA2 and APA3, theRAW264.7 cells were analyzed using a flow cytometer EPICS-ALTRA (BeckmanCoulter). As a result, as shown in FIG. 3, the RAW264.7 cells wererecognized by APA1 and APA2, but the cells were not recognized by APA3that is an antibody against the C-terminal region of the MMRP19 protein.

These results suggest that the MMRP19 protein consists of anextracellular domain consisting of about 98 amino acid residues at theN-terminus, a transmembrane (TM) domain consisting of about 30 aminoacid residues following the above domain, and an intracellular domainconsisting of about 113 amino acid residues further following the abovedomain.

Example 7 Association of the MMRP19 Protein with Induction of G-CSF GeneExpression

(1) Production of the Pica-RAW264.7 Cells

A PicaGene System (Wako Pure Chemical Industries, Ltd.) was used, inwhich a luciferase gene was used as a reporter gene. A G-CSF promotergene was inserted into the site from XboI to NcoI of a PicaGene EnhancerVector 2 (Wako Pure Chemical Industries, Ltd.), a luciferage gene wasligated downstream of the site instead of the G-CSF gene, and further aneomycin-resistance gene cut from pMC1Neo Poly A was inserted into theSalI site located downstream of SV40, so that a PicaGCSFneo vector wasconstructed.

Subsequently, the above vector was introduced into the mouse macrophagecell line RAW264.7 using the following method. The RAW264.7 cell line,which was in the logarithmic growth phase, was collected and washed oncewith an Eagle medium (EMEM) containing 10% fetal bovine serum (FBS:Bio-Whittacker) and nonessential amino acid (NEAA). The cells werewashed and resuspended in the same medium at a concentration of 2×10⁷cells/ml. The obtained cell suspension (250 μL, 5×10⁶ cells) was placedin a 0.4 cm cuvette and then mixed with 10 μg of PicaGCSFneo plasmid DNAwhich was purified by cesium chloride method. Then, using a Gene Pulser(Bio-Rad), high voltage pulse of 300 V and 960 μF was applied on themixture so as to introduce the vector into the cells.

Forty-eight hours after transformation, the obtained cells was treatedwith a medium containing 1 g/l geneticin (one type of neomycin), and 10to 15 days later, cells forming a colony was selected. Forty-three outof the 50 geneticin resistant clones were stimulated with LPS, and as aresult, significant increase of luciferase activity was observed. Ofthese clones, one clone showed extremely high luciferase activity, andthis was called RAW264.7 clone 27-3 (referred to also as “Pica-RAW264.7cell.”)

Whether or not luciferase activity reflects the actual G-CSF mRNAexpression was confirmed by the following experiment. That is to say,RAW264.7 clone 27-3 cells (1.5×10⁷) stimulated with LPS (10 μg/ml at afinal concentration) for 18 hours were washed with PBS. The cells weredissolved, and the total RNA was extracted therefrom. The total RNA waselectrophoresed on 1% formaldehyde agarose gel, and the total RNA wasthen transferred onto a nylon filter followed by Northern blot analystsusing the ³²P labeled mouse G-CSF cDNA as a probe. As a control, β-actinwas used. As a result, it was shown that the induction of the G-CSF mRNAcorrelates with the luciferase activity, and therefore it was confirmedthat luciferase activity reflects the actual G-CSF mRNA expression.

(2) Detection of Induction of G-CSF Gene Expression

The Pica-RAW264.7 cells were inoculated on a 96-well microplate so thatthe number of cells became 5×10⁴ cells/100 μl per well, and the cellswere cultured in an KMRM medium containing 10% FBS at 37° C. in thepresence of 5% CO₂. The anti-peptide antibodies (APA1, APA2 and APA3)and the 3-4H7 antibody described in Example 6 were added to the plate atdifferent concentrations, and the cells were stimulated overnight at 37°C. in the presence of 5% CO₂. These plates were washed three times witha PBS buffer, and the cells were dissolved with a PicaGene resolvent andthen subjected to centrifugal separation. The luciferase activity of theobtained supernatant sample was determined with a Luminometer CT-9000DChemoluminescence Determination Microplate Reader (Dia-Iatron Co.,Ltd.). The results are shown in FIG. 4.

The 3-4H7 antibody at a concentration of 77 pmol/ml showed approximately60-fold increase in G-CSF induction. As is the case with the 3-4H7antibody, the anti-peptide antibodies (APA1 and APA2) recognizing theextracellular region also showed increase in G-CSF induction to thePica-RAW264.7 cells (APA1: 24-fold, APA2: 21-fold). In contrast, theAPA3 antibody recognizing the intracellular region showed no increase inG-CSF induction. From these results, it was shown that APA1 and APA2 arebound to the extracellular region of the MMRP19 protein and they areassociated with the induction of G-CSF gene expression.

Example 8 Relationship Between Induction of G-CSF Gene Expression andSecretion by Stimulation by 3-4H7 Antibody in the Macrophage Cell Lines

The RAW264.7 or Pica-RAW264.7 cells were suspended in an EMEM mediumcontaining 10% FBS (1.2×10⁵ cells/ml), the cells of 90 μl per well(1×10⁴ cells/well) were inoculated on a 96-well microplate, and thenthey were subjected to preincubation overnight at 37° C. in the presenceof 5% CO₂. Thereafter, 10 μl of the 3-4H7 antibody or LPS solution atvarious concentrations was added to the each well, and it was stimulatedfor 24 hours at 37° C. in the presence of 5% CO₂. Thereafter, theculture supernatant was placed in a sample tube and it was followed bycentrifugal separation, and the supernatant was used for the followingbioassay system detecting G-CSF using the NFS-60 cells. Detection ofG-CSF by bioassay-was carried out as follows: That is, first, the NFS-60cells were washed three times with PBS, and then the cells weresuspended in an RPMI 1640 medium containing 5% FBS and 100 μM NEAA sothat the density of cells became 3×10⁵ cells/ml. Fifty μl each of theobtained NFS-60 cell suspension was inoculated on a 96-well microplate(1.5×10⁴ cells/well), preincubation was carried out overnight at 37° C.in the presence of 5% CO₂. Thereafter, a medium containing 50 μl of theculture supernatant of the RAW264.7 or Pica-RAW264.7 cells or a mediumcontaining G-CSF was added to each well and it was followed byincubation for 24 hours at 37° C. in the presence of 5% CO₂. Thereafter,10 μl of a WST solution (5 mM2-(4-iodophenyl9-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium-Na,0.2 mM 1-methoxy-5-methylphenazinium methylsulfide, 20 mM Hepes, pH 7.4)was added to each well and it was followed by incubation at 37° C. for 4hours. Growth of the NFS-60 cells was assayed by determining theabsorbance at 450 nm, using 650 nm as a reference wave length, and theamount of G-CSF contained in the medium was assayed, using thecalibration curve prepared using the cell growth induced by G-CSF as areference. Moreover, as with in the case Example 7, the luciferaseactivity of the Pica-RAW264.7 cells was analyzed. As a result, as shownin FIGS. 5 to 8, it was shown that the 3-4H7 antibody, stimulates theRAW264.7 cells and the Pica-RAW264.7 cells, which is similar to LPS, andit conc ntration-dependently promotes the secretion of G-CSF. Since thesecretion of G-CSF-required a lower concentration of the 3-4H7 antibodythan that for the induction of the G-CSF gene, it was suggested that thebiosynthesis and secretion of G-CSF requires only a small level of G-CSFgene induction.

Example 9 Epitopic Region of Antigen Molecule MMRP19 Recognized by 3-4H7Antibody

Synthesis of peptides corresponding to the extracellular domains of theMMRP19 protein, MMRP19 (12-25, P1), MMRP19 (19-36, P2), MMRP19 (32-46,P3), MMRP19 (41-55, P4), MMRP19 (50-62, P5), MMRP19 (57-71, P6), MMRP19(64-81, P7), MMRP19 (72-87, P8) and MMRP19 (81-98, P9) was carried outby 9-Fluorenylmethoxycarbonyl method, using a 433 Automated PeptideSynthesizer from Applied Biosystems. The synthesized peptide resin wastreated with trifluoroacetic acid, and the treated product was purifiedusing a reverse phase chromatography (Shimadzu LC8A) so as to obtainapproximately 25 mg each of peptides. Each (0.5 μg) of the nine peptides(P1 to P9) corresponding to the MMRP19 extracellular domains wasimmobilized on a each well of a 96-well microplate, and then it wasblocked with skim milk, and followed by washing three times with PBS. Tothis microplate, 50 μl of the 3-4H7 antibody solution was added (10μg/ml at the final concentration), and the mixture was incubated at 37°C. for 1 hour. This was three times washed with PBS containing 0.05%Tween-20, and then a peroxidase labeled anti-mouse IgM rat antibodysolution was added thereto and allowed to incubate them at 37° C. for 1hour. The reaction product was further washed five times with PBScontaining 0.05% Tween, and then a peroxidase substrate ABTS andhydrogen peroxide were added thereto and allowed to incubate them at 37°C. for 30 minutes. Thereafter, the absorption at 405 nm was determinedusing a Microplate Reader M-Tmax (Molecular Device). As a result, asshown in FIG. 9, only the peptides corresponding to positions 72 to 87(P8) and 81 to 98 (P9) of the MMRP19 showed a binding ability to the3-4H7 antibody, and the remaining peptides had almost no bindingactivity. These results show that the epitope of the MMRP19 to the 3-4H7antibody exists in the positions 72 to 98 of the MMRP19 protein.

Example 10 Induction Pattern of Cytokine by 3-4H7 Antibody

Pica-RAW264.7 cells were inoculated on a 96-well microplate at 5×10⁴cells/100 μl per well. The cells were cultured in an EMEM mediumcontaining 10% FBS at 37° C. in the presence of 5% CO₂. Thereafter, thecells were stimulated overnight with the 3-4H7 antibody (a finalconcentration at 60 μg/ml) or LPS (a final concentration at 100 ng/ml).After stimulation, the supernatant was collected and subjected to themeasuring concentrations of various cytokines by ELISA. At the sametime, the luciferase activity of the supernatant was determined so as toanalyze the induction of G-CSF gene expression. As a control, a mediumwithout neither the antibody nor LPS was used. Determination of IL-1α,IL-1β, IL-6, TNF-α and GM-CSF was carried out using a commerciallyavailable ELISA kit (Endogen). As a result, as shown in Table 3, the3-4H7 antibody showed relatively high induction of G-CSF gene expression(approximately 60-fold). However, the level of inducing other cytokineswas low (approximately 5.4-fold for IL-6, approximately 5.1-fold forTNF-α, significant change could not be detected for IL-1α, IL-1β andGM-CSF.) In contrast. LPS significantly induced not only G-CSF but alsoIL-1α, IL-β, IL-6, TNF-α and GM-CSF.

TABLE 3 Induction of G-CSF and other cytokines G-CFS IL-lα IL-lβ IL-6TNF-α GM-CSF 3-4H7 mAb 57.8 ± 7.1 0.7 ± 0.2 2.2 ± 1.5  5.4 ± 0.5 5.1 ±0.4  1.9 ± 0.4 (60 μg/ml) LPS 496.5 ± 13.7 93.6 ± 5.6  30.7 ± 8.7 1274.7 ± 410.0 475.7 ± 181.8 113.6 ± 21.8 (100 ng/ml) All values arebased on the control value which is defined as 1.

Example 11 Expression of the Human Counterpart of the MMR19 cDNA inMonkey-derived Cell Line COS7

A purified human counterpart of the MMR19 cDNA fragment was ligated to aplasmid vector (pCMV-Script), and the obtained recombinant vector(pCMV-Script MMR19) was transfected to the Escherichia coli XL10-Gold byheat shock. The recombinant vector produced from the Escherichia coliXL10-Gold was purified, and approximately 20 μg of the recombinantvector was transfected into monkey-derived cell line COS7 (1×10⁷ cells),which are the non-expression cells of the MMRP19 protein, by highvoltage pulse method. Then, the cells into which the recombinant vectorwas introduced were cultured in a Dulbecco's modified Eagle's medium fortwo days and incubated with the antibody (3-4H7) at 4° C. for 1 hour.Thereafter, the reacted cells were stained with the FITC-labeled secondantibody and then analyzed with a flow cytometer EPICS-ALTRA.(BeckmanCoulter).

As a result, as shown in FIG. 10, the 3-4H7 was bound to the cells inwhich the recombinant vector was introduced, and therefore it was foundthat as in the case of the mouse MMRP19 protein, a human counterpart ofthe MMRP19 protein expresses on the surface of cells.

Example 12 Excessive Expression of the MMRP19 Protein on the RAW264.7Cell Membrane

First, purified MMR19 cDNA was integrated into the restriction enzymeScaI site of an expression vector pCMV-Script (Stratagene) using DNAligase, so that an MMR19 expression vector was prepared. Theintroduction of the expression vector into the RAW264.7 cell was carriedout by high voltage pulse method (electroporation). That is to say,2×10⁷ of the RAW264.7 cells were suspended in 500 μl of potassiumphosphate buffer, in which 10 μg of the vector was dissolved, and thenthe suspension was poured in a cuvette. A high voltage pulse of 500 μFand 300 V was applied thereon using Gene Pulser (Biorad), and therebymaking a small pore on the cell membrane for a short time andintroducing the vector into the cells. Subsequently, after applying thepulse, the cells were inoculated in a 250 ml flask and then cultured ina 10% FBS-EMEM medium at 37° C. for 72 hours. Thereafter, geneticin wasadded to the culture medium at a final concentration of 1 g/l, the cellswere further cultured for two weeks, and the transformed cells resistantto geneticin were selected. Further, cells reacting to the 3-4H7antibody were selected twice from the obtained transformed cells by flowcytometry, so that the RAW264.7 (OE-RAW264.7) cells excessivelyexpressing the MMRP19 protein were obtained.

The flow cytometric analysis of the MMRP19 protein expressed on the cellmembrane of the RAW264.7 or OE-RAW264.7 cells was carried out asfollows: That is to say, the RAW264.7 or OE-RAW264.7 cells were preparedat 1.5×10⁶ cells in 100 μl of PBS, and then the 3-4H7 antibody was addedthereto (final concentration 5 μg/100 μl) followed by reaction at 4° C.for 1 hour. The reaction product was washed three times with PBScontaining 5% FBS and 0.05% NaN₃ by centrifugal separation, then an FITCfluorescent labeled anti-mouse IgM antibody was added to the cellsuspension at a final concentration of 3 μg/100 μl, and the mixture wasfurther reacted at 4° C. for 1 hour in shade. Thereafter, the cells werewashed five times with PBS containing 5% FBS and 0.05% NaN₃ bycentrifugal separation, and the fluorescent labeled cells were detectedand analyzed by flow cytometry (EPICS ALTRA, Beckman Coulter). As aresult, as shown in FIG. 11, it was shown that when compared with theRAW264.7 cells, the OE-RAW264.7 cells express an excessive amount of theMMRP19 protein on the cell membrane, and it was found that the MMRP19was expressed as a cell membrane prot in.

Example 13 Study of the Effect of MMRP19 Protein for ExcessiveExpression of G-CSF Gene Induction by 3-4H7 Antibody Stimulation inMacrophage Cells

The RAW264.7 cells, the OE-RAW264.7 cells of the MMRP19 proteinexcessive expression cells, were inoculated on a 6-well microplate, sothat the number of cells for each well became 1.5×10⁶ cells (5×10⁵cells/ml), and the cells were cultured overnight at 37° C. in thepresence of 5% CO₂. The 3-4H7 antibody (at final concentration of 50μg/ml) or LPS (at final concentration of 100 ng/ml) was added to theculture and allowed to stimulate the G-CSF gene induction at 37° C. inthe presence of 5% CO₂ for 3, 6, 9 or 12 hours. Then, the total RNA wasextracted from the RAW264.7 cells or OERAW264.7 cells stimulated withthe 3-4H7 antibody or LPS as described above by GuanidiumThiocyanate/Phenol Chloroform Extraction, and using reversetranscriptase (MMLV-RTase) and DNA polymerase, cONA was synthesized fromthe total RNA. Thereafter, using a sense primer(5′-GCTGTGGCAAAGTGCACT-3′ (SEQ ID NO: 11)) corresponding to thepositions 121 to 138 of the mouse GCSF gene sequence and an antisenseprimer (5′-ATCTGCTGCCAGATGGTG-3′ (SEQ ID NO: 12)) corresponding to thepositions 537 to 520 of the same sequence, RT-PCR reaction was carriedout. As a result, as shown in FIG. 12, in the RAW264.7 cells,significant G-CSF mRNA-induction was observed at as many as 6 hoursafter stimulation with the 3-4H7 antibody, but in the OE-RAW264.7 cells,G-CSF mRNA induction had already been observed at 3 hours after thestimulation. In the case of stimulation by LPS, G-CSF mRNA was inducedat as many as 6 hours after the stimulation both in the RAW264.7 cellsand in the OE-RAW264.7 cells. Thus, it was shown that when stimulationwith the same concentration of 3-4H7 antibody is given to both types ofcells, G-CSF mRNA-induction occurs more quickly in the OE-RAW264.7 cellsthan in the RAW264.7 cells. However, in the case of stimulation withLPS, no significant difference was observed between the two types ofcells regarding G-CSF mRNA induction. From the above results, it wassuggested that the G-CSF gene is induced by the binding of the 3-4H7antibody to the MMRP19 protein. Moreover, it was also suggested that inthe RAW264.7 cells or OE-RAW264.7 cells, the signal transmitting routeregarding G-CSF induction by stimulation with the 3-4H7 antibody mightdiffer from the case of stimulation with LPS.

Example 14 Enzymatic Immunodetection of G-CSF Secretion by Stimulationwith 3-4H7 Antibody in Macrophage Cells and Study of the Effect of theMMRP19 Protein Excessive Expression

The RAW264.7 cells, or the OE-RAW264.7 cells with the MMRP19 proteinexcessive expression were inoculated in a 5 cm petri dish, so that thenumber of cells became 1.5×10⁶ cells/5 ml (3×10⁵ cells/ml), and thecells were cultured overnight at 37° C. in the presence of 5% CO₂. The3-4H7 antibody (final concentration at 50 μg/ml) was added to theculture that was followed by stimulation at 37° C. in the presence of 5%CO₂ for 9, 12, 18 or 24 hours, and 100 μl of the culture supernatant wascollected after the above stimulation times. The culture supernatant wastransferred onto a 96-well immunoplate (Nunc, MaxiSorp), and solidifiedat 4° C. overnight that was followed by washing three times with 400 μlof PBS containing 0.05% Tween 20. Thereafter, 100 μl of rabbit serum wasadded to each well, and the mixture was incubated at room temperaturefor 30 minutes so that the well was blocked, and then it was againwashed three times with 400 μl of PBS containing 0.05% Tween 20.Thereafter, 100 μl of an anti-mouse G-CSF goat antibody (R&D) which wasprepared at a concentration of 10 μg/ml was added thereto, and themixture was incubated at 4° C. overnight that was followed by washingfive times with 400 μl of PBS containing 0.05% Tween 20. Thereafter, 100μl of a biotinated anti-goat Ig rabbit antibody solution was furtheradded to each well of the plate, and the mixture was incubated at roomtemperature for 1 hour that was followed by washing three times with 400μl of PBS containing 0.05% Tween 20. Thereafter, 100 μl of anavidin-peroxidase solution was further added thereto, and the mixturewas incubated at room temperature for 30 minutes that was followed bywashing five times with 400 μl of PBS containing 0.05% Tween 20. Afterthat, 100 μl of a peroxidase substrate ABTS solution was added to eachwell and allowed to set at room temperature for 30 minutes, andthereafter the absorption at 405 nm was analyzed using a MicroplateReader M-Tmax (Molecular Device). As a result, as shown in FIG. 13, itwas shown that the 3-4H7 antibody promotes the secretion of the G-CSFtime-dependently in the RAW264.7 cells, or the OE-RAW264.7 cells withthe MMRP19 protein excessive expression cells, and that the activity issignificantly higher in the OE-RAW264.7 cells. From these results(including Examples 7, 8 and 13), it was suggested that the 3-4H7antibody binds to the MMRP19 protein existing on the surface of themacrophage-like cell line, RAW264.7 cell, and activates the protein soas to allow the protein to induce or promote the secrection of G-CSF.

Example 15 Functional Analysis of MMRP19 Protein Homolog in Human Cells

Human-derived cultured cells, HL-60 cells (Riken, The Institute ofPhysical and Chemical Research) were placed in an RPMI 1640 mediumcontaining 10% FBS so that the cell density became 1.5×10⁵ to 1.5×10⁶cells/ml and cultured at 37° C. in the presence of 5% CO₂.Differentiation of the HL-60 cells into neutrophil-like cells wascarried out by treating the cells with 500 μM dibutyryl cAMP (dbcAMP)for 3 days. Further, the differentiation of the HL-60 cells intomacrophage-like cells was carried out by treating the cells with 100μg/ml phorbol 12-myristate 13-acetate (PMA) for 3 days.

First, for undifferentiated HL-60 cells, the neutrophil-likedifferentiated HL-60 cells and the macrophage-like differentiated HL-60cells, the presence of the cell membrane surface antigen recognized bythe 3-4H7 antibody was examined by flow cytometry. As a result, it wasshown that surface antigen for the 3-4H7 mAb antibody is present only inthe macrophage-like differentiated HL-60 cells, but the antigen is notpresent in the undifferentiated or the neutrophil-like differentiatedHL-60 cells. Moreover, when the lysates of undifferentiated,neutrophil-like differentiated and macrophage-like differentiated HL-60cells were prepared, and the synthesis of a human counterpart of theMMRP19 protein was analyzed by Western blotting using the 3-4H7antibody, or when the gene was detected by RT-PCR, the same results wereobtained. From these results, it was shown that the human counterpart ofthe MMRP19 protein that is an antigen for a 3-4H7 antibody expresses,when the HL-60 cell that is a human cultured cell is differentiated intoa macrophage-like cell by PMA.

Subsequently, the activity of the 3-4H7 antibody to induce or promotethe secretion of G-CSF in the HL-60 cells was studied. That is,undifferentiated, neutrophil-like differentiated and macrophage-likedifferentiated HL-60 cells were stimulated with the 3-4H7 antibody at afinal concentration of 60 μg/ml at 37° C. for 18 hours in the presenceof 5% CO₂. Thereafter, G-CSF secreted in the culture supernatant wasdetected by G-CSF bioassay (Example 8) and enzyme immunoassay (Example14). Further, G-CSF contained in the cells at this time was detected byWestern blotting, and G-CSF mRNA was detected by RT-PCR (Example 13). Asa result, it was shown that G-CSF is secreted again only in the culturesupernatant from the macrophage-like diff rentiated HL-60 cells. Fromthe above results, it was suggested that the 3-4H7 antibody binds to thehuman-type MMRP19 protein and activates the protein so as to allow theprotein to induce or promote the secrection of G-CSF also in themacrophage-like differentiated HL-60 cells.

Example 16 Induction of G-CSF mRNA Expression by 3-4H7 Antibody(Anti-MMRP19 Antibody)

<Method>

(1) Action of 3-4H7 Antibody on Bone Marrow Cells and PeritonealMacrophage Cells from the Normal Mice

Using BALB/c mice (male, 7-week old), bone marrow cells from the femurand the shank as well as the peritoneal macrophage cells were collected.An appropriate amount of cold Hank's balanced salt solution was suckedin a 1 ml syringe with a 21G needle, and the tip of the needle wasthrust into the bone cavity of the extirpated femur and shank to collect2 mL of bone marrow cells into the tube. Moreover, an appropriate amountof cold Hank's balanced salt solution was sucked in a 1 ml syringe, theabdominal cavity was well washed, and cells suspended in PBS solutionwere collected. This operation was repeated twice, and peritonealmacrophages were collected. The thus collected bone marrow cells(1.5×10⁶ cells) and peritoneal macrophage cells (5×10⁵ cells) wereincubated in an incubator at 37° C. in the presence of 5% CO₂ for 18hours. After cells were incubated, the 3-4H7 antibody (1,2,5,10 and 20μg/ml) was added thereto, and the mixture was further cultured in anincubator at 37° C. in the presence of 5% CO₂ for 6 hours. A group byadding only the solvent was used as a control.

As a result, the amount of G-CSF mRNA in the peritoneal macrophage cellsincreased by approximately 6.5-fold in the presence of the 3-4H7antibody (10 μg/ml) (FIG. 14). The cells were simultaneously stimulatedwith 20 ng/ml LPS, and the amount of G-CSF mRNA in the peritonealmacrophage cells increased by approximately 3.6-fold (FIG. 14).Moreover, the amount of G-CSF mRNA in the bone marrow cells increased byapproximately 3.2-fold in the presence of the 3-4H7 antibody (10 μg/ml)(FIG. 14). The cells were simultaneously stimulated with 20 ng/ml LPS,and the amount of G-CSF mRNA in the bone marrow cells increased byapproximately 2.2-fold (FIG. 14).

(2) Action of the 3-4H7 Antibody on the Normal Mouse Liver-derivedKupffer Cell

A mouse was anesthetized with pentobarbital, and the abdomen was thenincised while trying not to damage the liver. The bowel was placed tothe right so as to expose the portal and the inferior vena cava, and aliver perfusion liquid was perfused from the portal vein by a peristapump so that the blood was removed from the liver. The liver was thentreated with a collagenase solution. The liver was extirpated, and theextirpated liver was gently dissolved with a 10% FCS/RPMI1640/penicillin-streptomycin medium followed by filtration with a meshand the supernatant was collected in a 50 ml centrifugation tube.Centrifugal separation was carried out under conditions at 550 rpm for 2minutes and at 4° C. to collect a supernatant. This operation wasrepeated twice. Centrifugal separation was carried out under conditionsat 1,500 rpm for 10 minutes and at 4° C. to eliminate the supernatant,and then 10 ml of an RPMI 1640 medium was added to the remaining cellblock to obtain a cell suspension solution. The cell suspension wasincubated in an incubator at 37° C. in the presence of 5% CO₂ for 1 hourand then it was washed three times with an RPMI 1640 medium to eliminatenon-adhesive cells other than Kupffer cells. The 3-4H7 antibody (10, 30μg/ml) or lipopolysaccharide (LPS, 1 μg/ml) was added to the preparedKupffer cells, and the mixture was cultured in an incubator at 37° C. inthe presence of 5% CO₂ for 1, 3 and 6 hours. A group to which only thesolvent was added was used as a control.

As a result, at 1 hour after stimulation by the 3-4H7 antibody (30μg/ml), the amount of G-CSF mRNA in the liver-derived Kupffer cellsincreased by approximately 5.2-fold (FIG. 15). The liver-derived Kupffercells were simultaneously stimulated with 1 μg/ml of LPS for 1 hour, andthe amount of G-CSF mRNA in the cells increased by approximately4.4-fold (FIG. 15).

(3) Action of 3-4H7 Antibody on the Normal Mouse PeripheralBlood-derived Macrophage Cells

An equivalent amount of 10% FCS/RPMI 1640/penicillin-streptomycin mediumwas added to the blood collected from the mouse heart to prepare a cellsuspension solution. Lymphoprep (Nycomed) was placed in a 15 mlcentrifugation tube, the cell suspension solution was gently placedthereon and it was followed by centrifugal separation at 2,300 rpm for20 minutes at 4° C. The intermediate layer was gently collected, and tothe collected layer of cell suspension solution, the medium was addedand fully mixed, followed by centrifugal separation at 2,500 rpm for 5minutes at 4° C. The supernatant was eliminated, the remaining cellswere dissolved, and an RPMI 1640 medium was added thereto, followed bycentrifugal separation at 1,500 rpm for 5 minutes at 4° C. Thisoperation was repeated twice, and the cells were well washed. The viablecell was counted after staining them with a trypan blue solution. Thecell suspension solution (2×10⁶ cells) was inoculated on a 24-holeplate, and the plate was incubated in an incubator at 37° C. in thepresence of 5% CO₂ for 1 hour. Thereafter, it was washed three timeswith a medium to eliminate non-adhesive cells. M-CSF (30 ng/ml) wasadded to each well and the mixture was cultured in an incubator at 37°C. in the presence of 5% CO₂ for 4 days. The 3-4H7 antibody (10, 30μg/ml) or LPS (1 μg/ml) was added to the prepared macrophage cells, andthe mixture was cultured in an incubator at 37° C. in the presence of 5%CO₂ for 15 minutes and 1 hour. A group to which only the solvent wasadded was used as a control.

As a result, the 3-4H7 antibody (10 μg/ml) increased the amount of G-CSFmRNA in the macrophage cells by approximately 4.6-fold (FIG. 16). Themacrophage cells were simultaneously stimulated with 1 μg/ml LPS for 1hour, and the amount of G-CSF mRNA in the cells increased byapproximately 5.4-fold (FIG. 16).

(4) Preparation of the Total RNA

Using an RNeasy Mini Kit (QIAGEN), the total RNA was extracted from thecollected cells. This operation was carried out at room temperature. Theextracted total RNA (200 μl) was incubated at 65° C. for 10 minutes,followed by cooling on ice.

(5) Determination of the Mouse G-CSF mRNA Amount

The above prepared mRNA (10 μl) and Taq Man EZ RT-PCR CORE REAGENTS(Perkin Elmer) were mixed on a 96-well reaction Plate (Perkin Elmer),and using ABI Prism 7700 (Applied Biosystems), the amount of the mouseG-CSF mRNA was quantitated (number of cycles: 40). Using GAPDH as aninternal standard, the amount of each mRNA was corrected. The usedprimers are as follows:

mG-CSF forward: CAGCAGACACAGTGCCTAAGC, (SEQ ID NO: 13) mG-CSF reverse:AGTTGGCAACATCCAGCTGAA, (SEQ ID NO: 14) mGAPDH forward:TGCACCACCAACTGCTTAG, and (SEQ ID NO: 15) mGAPDH reverse:GGATGCAGGGATGATGTTC. (SEQ ID NO: 16)

As a probe, SYBER GREEN® was used for mG-CSF, andVic-CAGAAGACTGTGGATGGCCCCTC-Tamura (SEQ ID NO: 17) was used for mGAPDH.

As a result, at 3 hours after the administration of the 3-4H7 antibody,the amount of G-CSF mRNA in the peritoneal macrophage cells increased byapproximately 9-fold of untreated cells (FIG. 17).

(6) Action of 3-4H7 Antibody on Cyclophosphamide-inducedMyelosuppressive Mouse

Twenty-four BALB/c mice (male, 9-week old) were intraperitoneallyadministered with cyclophosphamide (25 mg/ml) at a ratio of 10 ml/kg(250 mg/kg). From 3 days after the administration of cyclophosphamide,the mice were intraperitoneally administered with PBS (a solvent of3-4H7), a 3-4H7 antibody solution (0.42 mg/ml) and rhG-CSF (1 μg/ml)-ata ratio of 10 ml/kg once a day for 3 days. Moreover, the mice weresubcutaneously administered with 10 ml/kg rhG-CSF (1 μg/ml) once a day.Seven days later, the blood (about 40 μl) was collected from the ocularvein of each mouse, using a heparin blood collecting tube, and thenumber of blood cells was counted with Sysmex F-800 (Sysmex). Moreover,a blood smear was prepared from the same blood, and it was subjected toMay-Grünwald Giemsa stain. After staining. 5 classes of 200 leukocyteswere counted with a microscope. For detection of significant difference,the implementation of variance analysis was confirmed by 1-way ANOVA,and then LSD test was carried out.

As a result, the number of leukocytes and neutrophils significantlyincreased on the 7^(th) day after the administration of cyclophosphamide(FIG. 18). When human G-CSF (10 mg/kg) as a positive control wasintraperitoneally administered to the mice for 3 days in the same manneras described above, the number of leukocytes and neutrophilssignificantly increased on the 7^(th) day after the administration, andthe increased amount was almost equivalent to the amount increased bythe administration of the 3-4H7 antibody (FIG. 18).

INDUSTRIAL APPLICABILITY

The inventive gene protein encoded by the gene (including a fragment ofthe above gene and a fragment of the above protein), antibody (includinga fragment thereof), receptor, and substance (including a low molecularsubstance) are novel, and these are useful as pharmaceuticals.

Moreover, the inventive gene, protein encoded by the gene (including afragment of the above gene and a fragment of the above protein),antibody (including a fragment thereof), receptor are also useful asanalytic reagents, when a substance capable of inducing and secreting aG-CSF (e.g. a monoclonal antibody, a protein, other low molecularsubstances, etc.) or the like is screened. Furthermore, since the G-CSFenhances the formation of erythroblasts by erythropoietin or theformation of blast cell colonies by interleukin-3, or promotes(reinforces and increases) blood cells such as leukocytes, erythrocytesor thrombocytes, the above substances Inducing the G-CSF can be used asagents promoting the generation of the G-CSF or agents controlling thebiological activity regarding the factor. Specifically, it is expectedthat the above substances can be used for treatment, diagnosis andothers of neutropenia, aplastic anemia and/or cytopenia regarding thereduction of the number of leukocytes, erythrocytes, thrombocytes or theothers, while preventing the side effect caused by the directadministration of the G-CSF as an agent.

In addition, the gene fragment of the present invention is useful alsoas a probe for screening of homologue genes derived from otherorganisms.

1. An isolated gene encoding a protein having the amino acid sequenceshown in SEQ ID NO:
 2. 2. An isolated gene having the nucleotidesequence shown in SEQ ID NO:
 1. 3. An isolated gene encoding (a) aprotein having the amino sequence shown in SEQ ID NO: 4; or (b) aprotein having at least 95% identity with the amino acid sequence shownin SEQ ID NO: 4 through the conservative substitution of one or moreacids and also binding to an antibody or an antibody fragment that isproduced by the hybridoma cell line deposit as FERM BP-6103.
 4. Anisolated gene having (a) the nucleotide sequence shown in SEQ ID NO: 3;(b) a nucleotide sequence encoding a protein having at least 95%identity with the amino acid sequence shown in SEQ ID NO: 4 through theconservative substitution of one or more amino acids and also binding toan antibody or an antibody fragment that is produced by the hybridomacell line deposited as FERM BP-6103; or (c) a nucleotide sequencehybridizing with DNA hiving the nucleotide sequence shown in SEQ ID NO:3 under stringent conditions of 6×SSC, 5× Denhardt's solution, 0.5% SDS,25-68° C. or 0-50% formamide, 6×SSC, 0.5% SDS, 25-68° C. and whichencodes a protein that can bind to an antibody or antibody fragment thatis produced by the hybridoma cell line deposited as FERM BP-6103.
 5. Theisolated gene according to any one of claims 1 to 4, which is a mousegene or a human gene.
 6. An isolated gene comprising any one of thefollowing nucleotide sequences: (a) a nucleotide sequence consisting ofnucleotides 519 to 736, nucleotides 666 to 689, nucleotides 381 to 403,or nucleotides 709 to 727 with respect to the nucleotide sequence shownin SEQ ID NO: 1; or (b) a nucleotide sequence having at least 95%identity with the nucleotide sequence of (a) above, and also encoding aprotein that binds to an antibody or an antibody fragment that isproduced by the hybridoma cell line deposited as FERM BP-6103.
 7. Apurified protein having the amino acid sequence shown in SEQ ID NO: 2.8. Any of the following purified proteins: (a) a protein having theamino acid sequence shown in SEQ ID NO: 4; (b) a protein having at least95% identity with the amino acid sequence shown in SEQ ID NO: 4 throughthe conservative substitution of one or more amino acids and alsobinding to an antibody or an antibody fragment that is produced by thehybridoma cell line deposited as FERM BP-6103; or (c) a protein encodedby DNA which hybridizes with DNA having the nucleotide sequence shown inSEQ ID NO: 3 under stringent conditions of 6×SSC, 5× Denhardt'ssolution, 0.05% SDS, 2.5-68° C. or 0-50% formamide, 6×SSC, 0.5% SDS,25-68° C. and which encodes a protein unit can bind to an antibody orantibody fragment that is produced by the hybridoma cell line depositedas FERM BP-6103.
 9. The purified protein according to claim 7 or 8,which is mouse or human protein.
 10. A purified protein having any oneof the following amino acid sequences: (a) an amino acid sequenceconsisting of amino acids 1 to 91, amino acids 50 to 146, ammo acids 1to 78, amino acids 200 to 241, amino acids 172 to 241, amino acids 103to 150, or (b) an amino acid sequence having at least 95% identity withthe amino acid sequence of (a) above, and also binding to an antibody oran antibody fragment that is produced by the hybridoma cell linedeposited as FERM BP-6103.
 11. A recombinant vector comprising the geneaccording to any one or claims 1 to
 4. 12. A transformed cell comprisinga recombinant vector that contains the gene according to any one ofclaims 1 to
 4. 13. An isolated receptor for a substance that can inducethe production of a granulocyte colony-stimulating factor whichcomprises a monoclonal antibody or an antibody fragment produced fronthybridoma cells deposited under Accession No. FERM BP-6103, wherein saidreceptor comprising the protein according to any one of claims 7 or 8,and is present in a cell which can produce granulocytecolony-stimulating factor.
 14. A method of screening for a substancethat can bind to the protein of claim 7 or 8, which comprises i)providing a potential substance; ii) exposing the potential substance tosaid protein, and iii) testing for specific bindine.
 15. A method ofscreening for a substance that can bind to the receptor according toclaim 13, which comprises i) providing a potential substance; ii)exposing the potential substance to said receptor, and iii) testing forspecific binding.